EP3475427A1 - Procédés et compositions destinés à être utilisés dans la modification du génome de plantes - Google Patents

Procédés et compositions destinés à être utilisés dans la modification du génome de plantes

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Publication number
EP3475427A1
EP3475427A1 EP17821073.8A EP17821073A EP3475427A1 EP 3475427 A1 EP3475427 A1 EP 3475427A1 EP 17821073 A EP17821073 A EP 17821073A EP 3475427 A1 EP3475427 A1 EP 3475427A1
Authority
EP
European Patent Office
Prior art keywords
sequence
interest
vector
strand
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP17821073.8A
Other languages
German (de)
English (en)
Other versions
EP3475427A4 (fr
Inventor
Elysia K. Krieger
Richard J. LAWRENCE
Zarir E. VAGHCHHIPAWALA
Jianping Xu
Xudong Ye
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Monsanto Technology LLC
Original Assignee
Monsanto Technology LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Monsanto Technology LLC filed Critical Monsanto Technology LLC
Publication of EP3475427A1 publication Critical patent/EP3475427A1/fr
Publication of EP3475427A4 publication Critical patent/EP3475427A4/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
    • C12N15/8205—Agrobacterium mediated transformation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
    • C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination

Definitions

  • This disclosure relates to the field of plant transformation.
  • Methods of, and compositions for, transfer of T-strands of DNA from Agrobacterium plasmids are provided.
  • methods of, and compositions for, using Agrobacterium-mediated transfer of T-strands of DNA to promote templated gene editing and site-directed integration of transgenes are also provided.
  • Agrobacterium-mediated transformation utilizes Agrobacterium tumefaciens to transfer single-stranded DNA synthesized from recombinant plasmids to plant cells.
  • Transformation of plant cells often requires co-culturing with tumefaciens.
  • a DNA sequence of interest is incorporated into specially-constructed DNA plasmids where the DNA sequence of interest is flanked by an Agrobacterium tumor-inducing (Ti) plasmid right border DNA region and a left border DNA region.
  • Ti Agrobacterium tumor-inducing
  • the Agrobacterium-mediated transformation process initiates when an endonuclease, VirD2, nicks the DNA plasmid at the right border and left border regions to release a single-stranded transfer DNA (also called the T-strand).
  • the T-strand transfers transgenes situated between the right and left borders into the targeted plant cells, where the T-strand can integrate into the genome (see, for example, U.S. Pat. No.
  • Plant cells that have been transformed via Agrobacterium-mediated transformation can be manipulated to regenerate into a whole, fertile plant.
  • Successful Agrobacterium-mediated transformation of a plant cell typically results in a random integration in the plant genome. Such random integrations can have deleterious effects to the plant cell if the transgene inserts into an essential endogenous gene. Similarly, such random integrations can have a deleterious effect on expression of the transgene due to position effects of the chromosome where the transgene inserted.
  • An Agrobacterium-mediated transformation method for plant cells that promotes templated gene editing and site-directed integration of transgenes.
  • the present disclosure provides a method of transforming a plant cell, comprising contacting the plant cell with a Rhizobiales cell capable of transforming the plant cell, where the Rhizobiales cell comprises at least one vector capable of forming two T- strands that are essentially complementary in at least a portion of the T-strands.
  • the at least one vector comprises a first right border DNA sequence (RBI), a second right border DNA sequence (RB2), and at least one sequence of interest
  • the RBI is positioned in the vector to initiate synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand
  • the RB2 is positioned in the vector to initiate synthesis of a second T-strand such that the sequence of interest is in the anti-sense orientation relative to the sequence of interest in the first T-strand, and the two T-strands resulting from initiation at RB 1 and RB2 are essentially
  • the at least one vector comprises a RBI, a RB2, a sequence of interest, a first left border DNA sequence (LB1) and a second left border DNA sequence (LB2), where the vector is configured such that the RBI is paired with the LB1 which are positioned in the vector to initiate (RBI) and terminate (LB 1) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the RB2 is paired with the LB2 which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti- sense orientation relative to the sequence of interest in the first T-strand, and where the two T-strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the sequence of interest.
  • the vector comprises a first sequence of interest and a second sequence of interest, where the first sequence of interest is essentially identical to the second sequence of interest, where the vector further comprises a RBI and a LBl which are positioned in the vector to initiate (RB I) and terminate (LBl) synthesis of a first T-strand such that the first sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand, and the vector further comprises a RB2 and a LB2 which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the second sequence of interest is in an anti-sense orientation relative to the first sequence of interest in the first T-strand, and where the two T-strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the first sequence of interest and the second sequence of interest.
  • the Rhizobiales cell comprises at least a first vector and a second vector, where each vector comprises essentially identical sequences of interest, and where the first vector comprises a RBI and a LB l which are positioned in the first vector to initiate (RBI) and terminate (LB 1) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and where the second vector comprises a RB2 and a LB2) which are positioned in the second vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti- sense orientation relative to the sequence of interest in the first T-strand, and where the two T-strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the sequence of interest.
  • the vector comprises a first sequence of interest, a second sequence of interest different from the first sequence of interest, at least two RB DNA sequences, and one or more optional LB DNA sequences, wherein the first RB DNA sequence (RBI) and a first LB DNA sequence (LBl) are positioned in the vector to initiate (RBI) and terminate (LBl) synthesis of the first T-strand such that the first sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the vector configuration further comprises a second RB DNA sequence (RB2) and a second LB DNA sequence (LB2) which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the first sequence of interest is in an anti-sense orientation from the 5' to 3' end of the second T-strand, wherein the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other in at least a portion of the first sequence of interest, and wherein the vector configuration
  • the instant disclosure provides a method of transforming a plant cell, comprising contacting the plant cell with two or more Rhizobiales cells capable of transforming the plant cell, where the two or more Rhizobiales cells each contain one of at least two vectors capable of forming two essentially complementary T-strands.
  • each vector comprises an essentially identical sequence of interest
  • the first vector comprises a first right border DNA sequence (RBI)
  • the RBI is positioned in the vector to initiate synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand
  • the second vector comprises a second right border DNA sequence (RB2) which is positioned in the vector to initiate synthesis of a second T-strand such that the sequence of interest is in the anti-sense orientation relative to the sequence of interest in the first T-strand, and where the two T- strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the sequence of interest.
  • each vector comprises an essentially identical sequence of interest
  • the first vector comprises a RBI and a first left border DNA sequence (LB1) which are positioned in the first vector to initiate (RB I) and terminate (LB 1) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand
  • the second vector comprises a RB2 and a second left border DNA sequence (LB2) which are positioned in the second vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation from the 5' to 3' end of the second T-strand, and where the sequence of interest in the two T-strands resulting from initiation at RB 1 and RB2 are essentially complementary to each other.
  • RBI is 5' to LB2.
  • RBI is 3' to LB2.
  • RB2 is 5' to LB 1.
  • RB2 is 3' to
  • the instant disclosure provides a method of increasing the rate of site directed integration of a sequence of interest, comprising contacting a plant cell with at least one vector capable of forming two essentially complementary T-strands.
  • the at least one vector comprises a first right border DNA sequence (RBI), a second right border DNA sequence (RB2), and at least one sequence of interest, where the RBI is positioned in the vector to initiate synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the RB2 is positioned in the vector to initiate synthesis of a second T-strand such that the sequence of interest is in the anti-sense orientation relative to the sequence of interest in the first T-strand, and where the two T-strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the sequence of interest.
  • the at least one vector comprises a RBI, a RB2, a sequence of interest, a first left border DNA sequence (LB1), a second left border DNA sequence (LB2) and where the vector is configured such that the RBI is paired with the LB1 which are positioned in the vector to initiate (RBI) and terminate (LB 1) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the RB2 is paired with the LB2 which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti- sense orientation relative to the sequence of interest in the first T-strand, and where the sequence of interest in the two T-strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the sequence of interest.
  • the vector comprises a first sequence of interest and a second sequence of interest, where the first sequence of interest is essentially identical to the second sequence of interest; where the vector further comprises a RBI and a LB1 which are positioned in the vector to initiate (RBI) and terminate (LB1) synthesis of a first T-strand such that the first sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the vector further comprises a RB2 and a LB2 which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the second sequence of interest is in an anti-sense orientation relative to the first sequence of interest in the first T-strand, and where the two T-strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the first sequence of interest and the second sequence of interest.
  • the Rhizobiales cell comprises at least a first and a second vector, where each vector comprises essentially identical sequences of interest, and where the first vector comprises a RBI and a LB1 which are positioned in the first vector to initiate (RBI) and terminate (LB1) synthesis of a first T- strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the second vector comprises a RB2 and a LB2 which are positioned in the second vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation relative to the sequence of interest in the first T-strand, and where the two T-strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the sequence of interest.
  • the vector comprises a first sequence of interest, a second sequence of interest different from the first sequence of interest, at least two RB DNA sequences, and one or more optional LB DNA sequences, wherein the first RB DNA sequence (RBI) and a first LB DNA sequence (LB1) are positioned in the vector to initiate (RBI) and terminate (LB1) synthesis of the first T-strand such that the first sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the vector configuration further comprises a second RB DNA sequence (RB2) and a second LB DNA sequence (LB2) which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T- strand such that the first sequence of interest is in an anti-sense orientation from the 5' to 3' end of the second T-strand, wherein the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other in at least a portion of the first sequence of interest, and wherein the vector configuration further
  • the instant disclosure provides a method of increasing the rate of site directed integration of a sequence of interest, comprising contacting a plant cell with two or more Rhizobiales cells, where the two or more Rhizobiales cells each contain one of at least two vectors capable of forming two essentially complementary T-strands.
  • each vector comprises an essentially identical sequence of interest
  • the first vector comprises a first right border DNA sequence (RBI)
  • the RBI is positioned in the vector to initiate synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand
  • the second vector comprises a second right border DNA sequence (RB2) which is positioned in the vector to initiate synthesis of a second T-strand such that the sequence of interest is in the anti-sense orientation relative to the sequence of interest in the first T-strand, and where the two T- strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the sequence of interest.
  • each vector comprises an essentially identical sequence of interest
  • the first vector comprises a RBI and a first left border (LBl) which are positioned in the first vector to initiate (RB I) and terminate (LBl) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand
  • the second vector comprises a RB2 and a second left border (LB2) which are positioned in the second vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti- sense orientation from the 5' to 3' end of the second T-strand, and where the sequence of interest in the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other.
  • the Rhizobiales cell is selected from an Agrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp., a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp.
  • the Rhizobiales cell further contains a vector comprising at least one expression cassette, wherein the expression cassettes comprise a sequence encoding a protein involved in DNA repair, and wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • RBI is 5' to LB2.
  • RBI is 3' to LB2.
  • RB2 is 5' to LBl .
  • RB2 is 3' to LBl .
  • the instant disclosure provides a method of transforming a plant cell, comprising contacting the plant cell with a Rhizobiales cell capable of transforming the plant cell, where the Rhizobiales cell comprises at least one vector comprising a right border DNA sequence (RB) and a left border DNA sequence (LB) and where the vector comprises between the RB and LB: (i) a first sequence of interest in a sense orientation relative to the RB, (ii) a spacer, and (iii) a second sequence of interest in an anti-sense orientation relative to the RB, where the first sequence of interest and second sequence of interest are essentially complementary and after synthesis of the T-strand anneal to form a double-stranded DNA.
  • the Rhizobiales cell is selected from an Agrobacterium spp., a
  • the Rhizobiales cell further contains a vector comprising at least one expression cassette, wherein the expression cassettes comprise a sequence encoding a protein involved in DNA repair, and wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • the instant disclosure provides a method of increasing the rate of site directed integration of a double-stranded DNA into a genome of a plant cell, comprising contacting the plant cell with at least one vector comprising a right border DNA sequence (RB) and a left border DNA sequence (LB), where the vector comprises between the RB and LB: (i) a first sequence of interest in a sense orientation relative to the RB, (ii) a spacer, and (iii) a second sequence of interest in an anti-sense orientation relative to the RB, where the first sequence of interest and second sequence of interest are essentially complementary and after synthesis of the T-anneal, where the double-stranded DNA is integrated into a genome of a plant cell.
  • RB right border DNA sequence
  • LB left border DNA sequence
  • the instant disclosure provides an Agrobacterium cell comprising at least one vector that is capable of forming two essentially complementary T-strands.
  • the instant disclosure provides an Agrobacterium cell comprising at least one vector comprising a right border DNA sequence (RB) and a left border DNA sequence (LB) and where the vector comprises between the RB and LB: (i) a first sequence of interest in a sense orientation relative to the RB, (ii) a spacer, and (iii) a second sequence of interest in an anti-sense orientation relative to the RB, where the first sequence of interest and second sequence of interest are essentially complementary, and after synthesis of the T- strand anneal to form a double-stranded DNA.
  • RB right border DNA sequence
  • LB left border DNA sequence
  • the instant disclosure provides a method of transforming a plant genome, comprising contacting at least one plant cell on a co-culture medium for at least 2 days, with at least one Rhizobiales cell capable of transforming the plant cell, where the Rhizobiales cell comprises at least one vector capable of forming two essentially
  • the Rhizobiales cell is selected from an Agrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp., a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp.
  • the Rhizobiales cell further contains a vector comprising at least one expression cassette, wherein the expression cassettes comprise a sequence encoding a protein involved in DNA repair, and wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • the instant disclosure provides a method of transforming a plant genome, comprising contacting at least one plant cell on a co-culture medium for at least 2 days, with at least one Rhizobiales cell capable of transforming the plant cell, where the Rhizobiales cell comprises at least one vector comprising a right border (RB) DNA sequence and a left border (LB) DNA sequence and where the vector further comprises between the RB and LB DNA sequences: (i) a first sequence of interest in a sense orientation relative to the RB DNA sequence, and (ii) a spacer, and (iii) a second sequence of interest in an anti- sense orientation relative to the RB DNA sequence, where the first sequence of interest and second sequence of interest are essentially complementary, and after synthesis of the T-strand anneal to form a double-stranded DNA.
  • the Rhizobiales cell comprises at least one vector comprising a right border (RB) DNA sequence and a left border (LB) DNA sequence and where the vector further comprises between the RB and LB DNA sequences: (i
  • the Rhizobiales cell is selected from an Agrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp., a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp.
  • the Rhizobiales cell further contains a vector comprising at least one expression cassette, wherein the expression cassettes comprise a sequence encoding a protein involved in DNA repair, and wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • the instant disclosure provides a method of transforming a plant genome, comprising contacting at least one plant cell on a co-culture medium for at least 3 days, with at least one Rhizobiales cell capable of transforming the plant cell, where the Rhizobiales cell comprises at least one vector capable of forming two essentially complementary T-strands.
  • the Rhizobiales cell is selected from an Agrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp., a Phyllobactenum spp., a Rhizobium spp., and a Sinorhizobium spp.
  • the Rhizobiales cell further contains a vector comprising at least one expression cassette, wherein the expression cassettes comprise a sequence encoding a protein involved in DNA repair, and wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • the instant disclosure provides a method of transforming a plant genome, comprising contacting at least one plant cell on a co-culture medium for at least 3 days, with at least one Rhizobiales cell capable of transforming the plant cell, where the
  • Rhizobiales cell comprises at least one vector comprising a right border (RB) DNA sequence and a left border (LB) DNA sequence and where the vector further comprises between the RB and LB DNA sequences: (i) a first sequence of interest in a sense orientation relative to the RB DNA sequence, and (ii) a spacer, and (iii) a second sequence of interest in an anti- sense orientation relative to the RB DNA sequence, where the first sequence of interest and second sequence of interest are essentially complementary, and after synthesis of the T-strand anneal to form a double-stranded DNA.
  • RB right border
  • LB left border
  • the Rhizobiales cell is selected from an Agrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp., a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp.
  • the Rhizobiales cell further contains a vector comprising at least one expression cassette, wherein the expression cassettes comprise a sequence encoding a protein involved in DNA repair, and wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • a method of providing a sequence of interest to a plant cell comprising contacting the plant cell with a Rhizobiales cell capable of transforming the plant cell, wherein the Rhizobiales cell comprises at least one vector capable of forming two T-strands that are essentially complementary in at least a portion of the T-strands, wherein the at least one vector comprises a first right border DNA sequence (RBI), a second right border DNA sequence (RB2), and at least one sequence of interest, and wherein the RBI is positioned in the vector to initiate synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the RB2 is positioned in the vector to initiate synthesis of a second T-strand such that the sequence of interest is in the anti-sense orientation relative to the sequence of interest in the first T-strand, and wherein the two T-strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of
  • the RBI and the RB2 are essentially homologous. In some embodiments, the LB1 and the LB2 are essentially homologous. In some embodiments, the RBI and the RB2 are not essentially homologous. In some embodiments, the LB1 and LB2 are not essentially homologous. In some embodiments, at least one of the RBI and RB2 comprise an Agrobacterium Ti plasmid right border consensus DNA sequence. In some embodiments, the right border consensus DNA sequence is selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, at least one of the RBI and RB2 comprise a sequence selected from SEQ ID NOs: 1-13.
  • At least one of the RBI and RB2 comprise a sequence at least 80% identical to a sequence selected from SEQ ID NOs:4 and SEQ ID NO: 12.
  • at least one of the LB 1 and LB2 comprise an Agrobacterium Ti plasmid left border consensus DNA sequence.
  • the left border consensus DNA sequence is selected from SEQ ID NO: 23 or SEQ ID NO: 24.
  • at least one of the LB 1 and LB2 comprise a sequence selected from SEQ ID NOs: 14-20.
  • at least one of the LB 1 and LB2 comprise a sequence at least 80% identical to SEQ ID NO: 19.
  • the sequence of interest comprises one or more expression cassettes.
  • the sequence of interest comprises one or more sequences selected from: a gene, a portion of a gene, an intergenic sequence, an enhancer, a promoter, an intron, an exon, a sequence encoding a transcription termination sequence, a sequence encoding a chloroplast targeting peptide, a sequence encoding a mitochondrial targeting peptide, an insulator sequence, a sequence encoding an anti-sense RNA construct, a sequence encoding non-protein-coding RNA (npcRNA), a sequence encoding a recombinase, a sequence encoding a recombinase recognition site, a landing pad, an editing template, an expression cassette, a stack of two or more expression cassettes encoding transgenes, a sequence encoding a site-specific enzyme, a sequence encoding a site-specific enzyme target site, a sequence encoding a selection marker, a sequence encoding a cell factor that functions to increase DNA repair
  • the sequence of interest comprises a sequence encoding a site-specific enzyme target site 5' to an expression cassette and a sequence encoding a site-specific enzyme target site 3' to an expression cassette.
  • the sequence of interest does not comprise a homology arm DNA sequence.
  • the sequence of interest comprises at least one homology arm DNA sequence.
  • the sequence of interest comprises both a left homology arm DNA sequence and a right homology arm DNA sequence.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, and ii) a second sequence that is not positioned between the left homology arm DNA sequence and the right homology arm DNA sequence.
  • the sequence of interest comprises a sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites. In some embodiments, the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence that is not positioned between sequences encoding site-specific enzyme target sites.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence encoding the site-specific enzyme, where the second sequence is not positioned between sequences encoding site-specific enzyme target sites.
  • at least one homology arm DNA sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%), or at least 99% identical to a target sequence in the plant genome.
  • the target sequence in the plant genome is a genie sequence. In some embodiments, the target sequence in the plant genome is a non-genic sequence. In some embodiments, the sequence of interest flanked by homology arms comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a native gene of the plant cell. In some embodiments, the sequence of interest comprises a protein-coding sequence. In some embodiments, the sequence of interest comprises a non- protein-coding RNA.
  • the non-protein-coding RNA is selected from the group consisting of: a microRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), a small RNA (22-26 nt in length) and precursor encoding same, a heterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), a hairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA (ta-siRNA), a naturally occurring antisense siRNA (nat- siRNA), a tracer RNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or any combination thereof.
  • the site-specific enzyme is selected from a group consisting of an endonuclease, a recombinase, and a transposase.
  • the endonuclease is selected from a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasX nuclease, a CasY nuclease, and a Cpfl nuclease.
  • TALEN transcription activator-like effector nuclease
  • Argonaute a Cas9 nuclease
  • CasX nuclease a CasX nuclease
  • CasY nuclease a CasY nuclease
  • Cpfl nuclease a Cpfl nuclease
  • the Cas9 nuclease is selected from the group comprising Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4 nuclease.
  • the recombinase is a tyrosine recombinase attached to a DNA recognition motif, or a serine recombinase attached to a DNA recognition motif.
  • the tyrosine recombinase attached to a DNA recognition motif is selected from the group consisting of a Cre recombinase, a Flp recombinase, and a Tnpl recombinase.
  • the serine recombinase attached to a DNA recognition motif is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase.
  • the transposase is a DNA transposase attached to a DNA binding domain.
  • the sequence of interest further comprises at least one site-specific enzyme target site.
  • the at least one site-specific enzyme target site is selected from a Cre/lox recombination site, a Flp/FRT recombination site, a endonuclease recognition site, and a TALEN site.
  • the sequence of interest comprises a sequence encoding a protein involved in DNA repair, wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via homologous recombination, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via non-homologous end joining, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • the plant cell already comprises a site-specific enzyme.
  • the plant cell is selected from the group consisting of a corn cell, a soybean cell, a canola cell, a cotton cell, a wheat cell, or a sugarcane cell.
  • a nucleotide sequence encoding the site-specific enzyme is stably transformed into the plant cell.
  • the Rhizobiales cell is selected from an Agrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp., an
  • the Rhizobiales cell further contains a vector comprising at least one expression cassette, wherein the expression cassettes comprise a sequence encoding a protein involved in DNA repair, and wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • a method of providing a sequence of interest to a plant cell comprising contacting the plant cell with a Rhizobiales cell capable of transforming the plant cell, wherein the Rhizobiales cell comprises at least one vector capable of forming two T-strands that are essentially complementary in at least a portion of the T-strands, wherein the at least one vector comprises a RBI, a RB2, a sequence of interest, a first left border DNA sequence (LB1) and a second left border DNA sequence (LB2), wherein the vector is configured such that the RBI is paired with the LB1 which are positioned in the vector to initiate (RBI) and terminate (LB1) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the RB2 is paired with the LB2 which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation relative to the sequence
  • the RBI and the RB2 are essentially homologous. In some embodiments, the LB1 and the LB2 are essentially homologous. In some embodiments, the RBI and the RB2 are not essentially homologous. In some embodiments, the LB1 and LB2 are not essentially homologous. In some embodiments, at least one of the RBI and RB2 comprise an Agrobacterium Ti plasmid right border consensus DNA sequence. In some embodiments, the right border consensus DNA sequence is selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, at least one of the RBI and RB2 comprise a sequence selected from SEQ ID NOs: 1-13.
  • At least one of the RBI and RB2 comprise a sequence at least 80% identical to a sequence selected from SEQ ID NOs:4 and SEQ ID NO: 12.
  • at least one of the LBl and LB2 comprise an Agrobacterium Ti plasmid left border consensus DNA sequence.
  • the left border consensus DNA sequence is selected from SEQ ID NO: 23 or SEQ ID NO: 24.
  • at least one of the LBl and LB2 comprise a sequence selected from SEQ ID NOs: 14-20.
  • at least one of the LBl and LB2 comprise a sequence at least 80% identical to SEQ ID NO: 19.
  • the sequence of interest comprises one or more expression cassettes.
  • the sequence of interest comprises one or more sequences selected from: a gene, a portion of a gene, an intergenic sequence, an enhancer, a promoter, an intron, an exon, a sequence encoding a transcription termination sequence, a sequence encoding a chloroplast targeting peptide, a sequence encoding a mitochondrial targeting peptide, an insulator sequence, a sequence encoding an anti-sense RNA construct, a sequence encoding non-protein-coding RNA (npcRNA), a sequence encoding a recombinase, a sequence encoding a recombinase recognition site, a landing pad, an editing template, an expression cassette, a stack of two or more expression cassettes encoding transgenes, a sequence encoding a site-specific enzyme, a sequence encoding a site-specific enzyme target site, a sequence encoding a selection marker, a sequence encoding a cell factor that functions to increase DNA repair
  • the sequence of interest comprises a sequence encoding a site-specific enzyme target site 5' to an expression cassette and a sequence encoding a site-specific enzyme target site 3' to an expression cassette.
  • the sequence of interest does not comprise a homology arm DNA sequence.
  • the sequence of interest comprises at least one homology arm DNA sequence.
  • the sequence of interest comprises both a left homology arm DNA sequence and a right homology arm DNA sequence.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, and ii) a second sequence that is not positioned between the left homology arm DNA sequence and the right homology arm DNA sequence.
  • the sequence of interest comprises a sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites. In some embodiments, the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence that is not positioned between sequences encoding site-specific enzyme target sites.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence encoding the site-specific enzyme, where the second sequence is not positioned between sequences encoding site-specific enzyme target sites.
  • at least one homology arm DNA sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a target sequence in the plant genome.
  • the target sequence in the plant genome is a genie sequence.
  • the target sequence in the plant genome is a non-genic sequence.
  • the sequence of interest flanked by homology arms comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), or 100% identical to a native gene of the plant cell.
  • the sequence of interest comprises a protein-coding sequence.
  • the sequence of interest comprises a non-protein-coding RNA.
  • the non- protein-coding RNA is selected from the group consisting of: a microRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), a small RNA (22-26 nt in length) and precursor encoding same, a heterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), a hairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA (ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a tracer RNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or any combination thereof.
  • miRNA microRNA
  • miRNA precursor a small interfering RNA
  • siRNA small interfering RNA
  • small RNA 22-26 nt in length
  • precursor precursor encoding same
  • hc-siRNA a heterochromatic siRNA
  • the site-specific enzyme is selected from a group consisting of an
  • the endonuclease is selected from a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasX nuclease, a CasY nuclease, and a Cpfl nuclease.
  • TALEN transcription activator-like effector nuclease
  • the Cas9 nuclease is selected from the group comprising Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csfi, and Csf4 nuclease.
  • the recombinase is a tyrosine
  • the tyrosine recombinase attached to a DNA recognition motif is selected from the group consisting of a Cre recombinase, a Flp recombinase, and a Tnpl recombinase.
  • the serine recombinase attached to a DNA recognition motif is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase.
  • the transposase is a DNA transposase attached to a DNA binding domain.
  • the sequence of interest further comprises at least one site-specific enzyme target site.
  • the at least one site-specific enzyme target site is selected from a Cre/lox recombination site, a Flp/FRT recombination site, a endonuclease recognition site, and a TALEN site.
  • the sequence of interest comprises a sequence encoding a protein involved in DNA repair, wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via homologous recombination, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via non-homologous end joining, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • the Rhizobiales cell is selected from an Agrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp., a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp.
  • the Rhizobiales cell further contains a vector comprising at least one expression cassette, wherein the expression cassettes comprise a sequence encoding a protein involved in DNA repair, and wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant- negative Ku70, or any combination thereof.
  • the plant cell already comprises a site-specific enzyme.
  • the plant cell is selected from the group consisting of a corn cell, a soybean cell, a canola cell, a cotton cell, a wheat cell, or a sugarcane cell.
  • a nucleotide sequence encoding the site-specific enzyme is stably transformed into the plant cell.
  • a method of providing a sequence of interest to a plant cell comprising contacting the plant cell with a Rhizobiales cell capable of transforming the plant cell, wherein the Rhizobiales cell comprises at least one vector capable of forming two T-strands that are essentially complementary in at least a portion of the T-strands, wherein the vector comprises a first sequence of interest and a second sequence of interest, wherein the first sequence of interest is essentially identical to the second sequence of interest; wherein the vector further comprises a RB 1 and a LB 1 which are positioned in the vector to initiate (RB 1) and terminate (LBl) synthesis of a first T-strand such that the first sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the vector further comprises a RB2 and a LB2 which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the second sequence of interest is in an anti-sense orientation relative to the first sequence of interest in
  • the RBI and the RB2 are essentially homologous. In some embodiments, the LBl and the LB2 are essentially homologous. In some embodiments, the RBI and the RB2 are not essentially homologous. In some embodiments, the LBl and LB2 are not essentially homologous. In some embodiments, at least one of the RBI and RB2 comprise an
  • the right border consensus DNA sequence is selected from SEQ ID NO: 21 or SEQ ID NO: 22.
  • at least one of the RBI and RB2 comprise a sequence selected from SEQ ID NOs: 1-13.
  • at least one of the RBI and RB2 comprise a sequence at least 80% identical to a sequence selected from SEQ ID NOs:4 and SEQ ID NO: 12.
  • at least one of the LB l and LB2 comprise an Agrobacterium Ti plasmid left border consensus DNA sequence.
  • the left border consensus DNA sequence is selected from SEQ ID NO: 23 or SEQ ID NO: 24.
  • At least one of the LB l and LB2 comprise a sequence selected from SEQ ID NOs: 14-20. In some embodiments, at least one of the LBl and LB2 comprise a sequence at least 80% identical to SEQ ID NO: 19. In some embodiments, the sequence of interest comprises one or more expression cassettes.
  • the sequence of interest comprises one or more sequences selected from: a gene, a portion of a gene, an intergenic sequence, an enhancer, a promoter, an intron, an exon, a sequence encoding a transcription termination sequence, a sequence encoding a chloroplast targeting peptide, a sequence encoding a mitochondrial targeting peptide, an insulator sequence, a sequence encoding an anti-sense RNA construct, a sequence encoding non-protein-coding RNA (npcRNA), a sequence encoding a recombinase, a sequence encoding a recombinase recognition site, a landing pad, an editing template, an expression cassette, a stack of two or more expression cassettes encoding transgenes, a sequence encoding a site-specific enzyme, a sequence encoding a site-specific enzyme target site, a sequence encoding a selection marker, a sequence encoding a cell factor that functions to increase DNA repair
  • the sequence of interest comprises a sequence encoding a site-specific enzyme target site 5' to an expression cassette and a sequence encoding a site-specific enzyme target site 3' to an expression cassette.
  • the sequence of interest does not comprise a homology arm DNA sequence.
  • the sequence of interest comprises at least one homology arm DNA sequence.
  • the sequence of interest comprises both a left homology arm DNA sequence and a right homology arm DNA sequence.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, and ii) a second sequence that is not positioned between the left homology arm DNA sequence and the right homology arm DNA sequence.
  • the sequence of interest comprises a sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites. In some embodiments, the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence that is not positioned between sequences encoding site-specific enzyme target sites.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence encoding the site-specific enzyme, where the second sequence is not positioned between sequences encoding site-specific enzyme target sites.
  • at least one homology arm DNA sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a target sequence in the plant genome.
  • the target sequence in the plant genome is a genie sequence.
  • the target sequence in the plant genome is a non-genic sequence.
  • the sequence of interest flanked by homology arms comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), or 100% identical to a native gene of the plant cell.
  • the sequence of interest comprises a protein-coding sequence.
  • the sequence of interest comprises a non-protein-coding RNA.
  • the non- protein-coding RNA is selected from the group consisting of: a microRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), a small RNA (22-26 nt in length) and precursor encoding same, a heterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), a hairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA (ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a tracer RNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or any combination thereof.
  • miRNA microRNA
  • miRNA precursor a small interfering RNA
  • siRNA small interfering RNA
  • small RNA 22-26 nt in length
  • precursor precursor encoding same
  • hc-siRNA a heterochromatic siRNA
  • the site-specific enzyme is selected from a group consisting of an
  • the endonuclease is selected from a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasX nuclease, a CasY nuclease, and a Cpfl nuclease.
  • TALEN transcription activator-like effector nuclease
  • the Cas9 nuclease is selected from the group comprising Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4 nuclease.
  • the recombinase is a tyrosine
  • the tyrosine recombinase attached to a DNA recognition motif is selected from the group consisting of a Cre recombinase, a Flp recombinase, and a Tnpl recombinase.
  • the serine recombinase attached to a DNA recognition motif is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase.
  • the transposase is a DNA transposase attached to a DNA binding domain.
  • the sequence of interest further comprises at least one site-specific enzyme target site.
  • the at least one site-specific enzyme target site is selected from a Cre/lox recombination site, a Flp/FRT recombination site, a
  • the sequence of interest comprises a sequence encoding a protein involved in DNA repair, wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via homologous recombination, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via non-homologous end joining, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • the plant cell already comprises a site-specific enzyme.
  • the plant cell is selected from the group consisting of a corn cell, a soybean cell, a canola cell, a cotton cell, a wheat cell, or a sugarcane cell.
  • a nucleotide sequence encoding the site-specific enzyme is stably transformed into the plant cell.
  • the Rhizobiales cell is selected from an Agrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp., a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp.
  • the Rhizobiales cell further contains a vector comprising at least one expression cassette, wherein the expression cassettes comprise a sequence encoding a protein involved in DNA repair, and wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant- negative Ku70, or any combination thereof.
  • a method of providing a sequence of interest to a plant cell comprising contacting the plant cell with a Rhizobiales cell capable of transforming the plant cell, wherein the Rhizobiales cell comprises at least one vector capable of forming two T-strands that are essentially complementary in at least a portion of the T-strands, wherein the Rhizobiales cell comprises at least a first vector and a second vector, wherein each vector comprises essentially identical sequences of interest, and wherein the first vector comprises a RBI and a LBl which are positioned in the first vector to initiate (RB I) and terminate (LBl) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and wherein the second vector comprises a RB2 and a LB2) which are positioned in the second vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation relative to the sequence of interest in the first
  • the RBI and the RB2 are essentially homologous. In some embodiments, the LBl and the LB2 are essentially homologous. In some embodiments, the RBI and the RB2 are not essentially homologous. In some embodiments, the LBl and LB2 are not essentially homologous. In some embodiments, at least one of the RBI and RB2 comprise an Agrobacterium Ti plasmid right border consensus DNA sequence. In some embodiments, the right border consensus DNA sequence is selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, at least one of the RBI and RB2 comprise a sequence selected from SEQ ID NOs: 1-13.
  • At least one of the RBI and RB2 comprise a sequence at least 80% identical to a sequence selected from SEQ ID NOs:4 and SEQ ID NO: 12.
  • at least one of the LBl and LB2 comprise an Agrobacterium Ti plasmid left border consensus DNA sequence.
  • the left border consensus DNA sequence is selected from SEQ ID NO: 23 or SEQ ID NO: 24.
  • at least one of the LBl and LB2 comprise a sequence selected from SEQ ID NOs: 14-20.
  • at least one of the LBl and LB2 comprise a sequence at least 80% identical to SEQ ID NO: 19.
  • the sequence of interest comprises one or more expression cassettes.
  • the sequence of interest comprises one or more sequences selected from: a gene, a portion of a gene, an intergenic sequence, an enhancer, a promoter, an intron, an exon, a sequence encoding a transcription termination sequence, a sequence encoding a chloroplast targeting peptide, a sequence encoding a mitochondrial targeting peptide, an insulator sequence, a sequence encoding an anti-sense RNA construct, a sequence encoding non-protein-coding RNA (npcRNA), a sequence encoding a recombinase, a sequence encoding a recombinase recognition site, a landing pad, an editing template, an expression cassette, a stack of two or more expression cassettes encoding transgenes, a sequence encoding a site-specific enzyme, a sequence encoding a site-specific enzyme target site, a sequence encoding a selection marker, a sequence encoding a cell factor that functions to increase DNA repair
  • the sequence of interest comprises a sequence encoding a site-specific enzyme target site 5' to an expression cassette and a sequence encoding a site-specific enzyme target site 3' to an expression cassette.
  • the sequence of interest does not comprise a homology arm DNA sequence.
  • the sequence of interest comprises at least one homology arm DNA sequence.
  • the sequence of interest comprises both a left homology arm DNA sequence and a right homology arm DNA sequence.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, and ii) a second sequence that is not positioned between the left homology arm DNA sequence and the right homology arm DNA sequence.
  • the sequence of interest comprises a sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites. In some embodiments, the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence that is not positioned between sequences encoding site-specific enzyme target sites.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence encoding the site-specific enzyme, where the second sequence is not positioned between sequences encoding site-specific enzyme target sites.
  • at least one homology arm DNA sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a target sequence in the plant genome.
  • the target sequence in the plant genome is a genie sequence.
  • the target sequence in the plant genome is a non-genic sequence.
  • the sequence of interest flanked by homology arms comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), or 100% identical to a native gene of the plant cell.
  • the sequence of interest comprises a protein-coding sequence.
  • the sequence of interest comprises a non-protein-coding RNA.
  • the non- protein-coding RNA is selected from the group consisting of: a microRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), a small RNA (22-26 nt in length) and precursor encoding same, a heterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), a hairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA (ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a tracer RNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or any combination thereof.
  • miRNA microRNA
  • miRNA precursor a small interfering RNA
  • siRNA small interfering RNA
  • small RNA 22-26 nt in length
  • precursor precursor encoding same
  • hc-siRNA a heterochromatic siRNA
  • the site-specific enzyme is selected from a group consisting of an
  • the endonuclease is selected from a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasX nuclease, a CasY nuclease, and a Cpfl nuclease.
  • TALEN transcription activator-like effector nuclease
  • the Cas9 nuclease is selected from the group comprising Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4 nuclease.
  • the recombinase is a tyrosine
  • the tyrosine recombinase attached to a DNA recognition motif is selected from the group consisting of a Cre recombinase, a Flp recombinase, and a Tnpl recombinase.
  • the serine recombinase attached to a DNA recognition motif is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase.
  • the transposase is a DNA transposase attached to a DNA binding domain.
  • the sequence of interest further comprises at least one site-specific enzyme target site.
  • the at least one site-specific enzyme target site is selected from a Cre/lox recombination site, a Flp/FRT recombination site, a endonuclease recognition site, and a TALEN site.
  • the sequence of interest comprises a sequence encoding a protein involved in DNA repair, wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via homologous recombination, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via non-homologous end joining, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • the plant cell already comprises a site-specific enzyme.
  • the plant cell is selected from the group consisting of a corn cell, a soybean cell, a canola cell, a cotton cell, a wheat cell, or a sugarcane cell.
  • a nucleotide sequence encoding the site-specific enzyme is stably transformed into the plant cell.
  • the Rhizobiales cell is selected from an Agrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp., a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp.
  • the Rhizobiales cell further contains a vector comprising at least one expression cassette, wherein the expression cassettes comprise a sequence encoding a protein involved in DNA repair, and wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant- negative Ku70, or any combination thereof.
  • a method of providing a sequence of interest to a plant cell comprising contacting the plant cell with a Rhizobiales cell capable of transforming the plant cell, wherein the Rhizobiales cell comprises at least one vector capable of forming two T-strands that are essentially complementary in at least a portion of the T-strands, wherein the at least one vector comprises a first sequence of interest, a second sequence of interest different from the first sequence of interest, at least two RB DNA sequences, and one or more optional LB DNA sequences, wherein the first RB DNA sequence (RBI) and a first LB DNA sequence (LBl) are positioned in the vector to initiate (RBI) and terminate (LBl) synthesis of the first T-strand such that the first sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the vector configuration further comprises a second RB DNA sequence (RB2) and a second LB DNA sequence (LB2) which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of
  • the RBI and the RB2 are essentially homologous. In some embodiments, the LBl and the LB2 are essentially homologous. In some embodiments, the RBI and the RB2 are not essentially homologous. In some embodiments, the LBl and LB2 are not essentially homologous. In some embodiments, at least one of the RBI and RB2 comprise an Agrobacterium Ti plasmid right border consensus DNA sequence. In some embodiments, the right border consensus DNA sequence is selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, at least one of the RBI and RB2 comprise a sequence selected from SEQ ID NOs: 1-13.
  • At least one of the RBI and RB2 comprise a sequence at least 80% identical to a sequence selected from SEQ ID NOs:4 and SEQ ID NO: 12.
  • at least one of the LBl and LB2 comprise an Agrobacterium Ti plasmid left border consensus DNA sequence.
  • the left border consensus DNA sequence is selected from SEQ ID NO: 23 or SEQ ID NO: 24.
  • at least one of the LBl and LB2 comprise a sequence selected from SEQ ID NOs: 14-20.
  • at least one of the LBl and LB2 comprise a sequence at least 80% identical to SEQ ID NO: 19.
  • the sequence of interest comprises one or more expression cassettes.
  • the sequence of interest comprises one or more sequences selected from: a gene, a portion of a gene, an intergenic sequence, an enhancer, a promoter, an intron, an exon, a sequence encoding a transcription termination sequence, a sequence encoding a chloroplast targeting peptide, a sequence encoding a mitochondrial targeting peptide, an insulator sequence, a sequence encoding an anti-sense RNA construct, a sequence encoding non-protein-coding RNA (npcRNA), a sequence encoding a recombinase, a sequence encoding a recombinase recognition site, a landing pad, an editing template, an expression cassette, a stack of two or more expression cassettes encoding transgenes, a sequence encoding a site-specific enzyme, a sequence encoding a site-specific enzyme target site, a sequence encoding a selection marker, a sequence encoding a cell factor that functions to increase DNA repair
  • the sequence of interest comprises a sequence encoding a site-specific enzyme target site 5' to an expression cassette and a sequence encoding a site-specific enzyme target site 3' to an expression cassette.
  • the sequence of interest does not comprise a homology arm DNA sequence.
  • the sequence of interest comprises at least one homology arm DNA sequence.
  • the sequence of interest comprises both a left homology arm DNA sequence and a right homology arm DNA sequence.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, and ii) a second sequence that is not positioned between the left homology arm DNA sequence and the right homology arm DNA sequence.
  • the sequence of interest comprises a sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites. In some embodiments, the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence that is not positioned between sequences encoding site-specific enzyme target sites.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence encoding the site-specific enzyme, where the second sequence is not positioned between sequences encoding site-specific enzyme target sites.
  • at least one homology arm DNA sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a target sequence in the plant genome.
  • the target sequence in the plant genome is a genie sequence.
  • the target sequence in the plant genome is a non-genic sequence.
  • the sequence of interest flanked by homology arms comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a native gene of the plant cell.
  • the sequence of interest comprises a protein-coding sequence.
  • the sequence of interest comprises a non-protein-coding RNA.
  • the non- protein-coding RNA is selected from the group consisting of: a microRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), a small RNA (22-26 nt in length) and precursor encoding same, a heterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA
  • RNA hairpin double strand RNA
  • ta-siRNA trans-acting siRNA
  • nat-siRNA naturally occurring antisense siRNA
  • tcRNA tracer RNA
  • gRNA guide RNA
  • sgRNA single-guide RNA
  • the site-specific enzyme is selected from a group consisting of an
  • the endonuclease is selected from a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasX nuclease, a CasY nuclease, and a Cpfl nuclease.
  • TALEN transcription activator-like effector nuclease
  • the Cas9 nuclease is selected from the group comprising Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4 nuclease.
  • the recombinase is a tyrosine
  • the tyrosine recombinase attached to a DNA recognition motif is selected from the group consisting of a Cre recombinase, a Flp recombinase, and a Tnpl recombinase.
  • the serine recombinase attached to a DNA recognition motif is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase.
  • the transposase is a DNA transposase attached to a DNA binding domain.
  • the sequence of interest further comprises at least one site-specific enzyme target site.
  • the at least one site-specific enzyme target site is selected from a Cre/lox recombination site, a Flp/FRT recombination site, a endonuclease recognition site, and a TALEN site.
  • the sequence of interest comprises a sequence encoding a protein involved in DNA repair, wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via homologous recombination, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via non-homologous end j oining, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • the Rhizobiales cell is selected from an Agrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp., a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp.
  • the Rhizobiales cell further contains a vector comprising at least one expression cassette, wherein the expression cassettes comprise a sequence encoding a protein involved in DNA repair, and wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant- negative Ku70, or any combination thereof.
  • the plant cell already comprises a site-specific enzyme.
  • the plant cell is selected from the group consisting of a corn cell, a soybean cell, a canola cell, a cotton cell, a wheat cell, or a sugarcane cell.
  • a nucleotide sequence encoding the site-specific enzyme is stably transformed into the plant cell.
  • Several embodiments relate to a method of transforming a plant cell, comprising contacting the plant cell with two or more Rhizobiales cells capable of transforming the plant cell, wherein the two or more Rhizobiales cells each contain one of at least two vectors capable of forming two essentially complementary T-strands, wherein each vector comprises an essentially identical sequence of interest, and where the first vector comprises a first right border DNA sequence (RBI), and wherein the RB I is positioned in the vector to initiate synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the second vector comprises a second right border DNA sequence (RB2) which is positioned in the vector to initiate synthesis of a second T- strand such that the sequence of interest is in the anti-sense orientation relative to the sequence of interest in the first T-strand, and wherein the two T-strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the sequence of interest.
  • the RBI and the RB2 are essentially homologous. In some embodiments, the LBl and the LB2 are essentially homologous. In some embodiments, the RBI and the RB2 are not essentially homologous. In some embodiments, the LBl and LB2 are not essentially homologous. In some embodiments, at least one of the RBI and RB2 comprise an Agrobacterium Ti plasmid right border consensus DNA sequence. In some embodiments, the right border consensus DNA sequence is selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, at least one of the RBI and RB2 comprise a sequence selected from SEQ ID NOs: 1-13.
  • At least one of the RBI and RB2 comprise a sequence at least 80% identical to a sequence selected from SEQ ID NOs:4 and SEQ ID NO: 12.
  • at least one of the LBl and LB2 comprise an Agrobacterium Ti plasmid left border consensus DNA sequence.
  • the left border consensus DNA sequence is selected from SEQ ID NO: 23 or SEQ ID NO: 24.
  • at least one of the LBl and LB2 comprise a sequence selected from SEQ ID NOs: 14-20.
  • at least one of the LBl and LB2 comprise a sequence at least 80% identical to SEQ ID NO: 19.
  • the sequence of interest comprises one or more expression cassettes.
  • the sequence of interest comprises one or more sequences selected from: a gene, a portion of a gene, an intergenic sequence, an enhancer, a promoter, an intron, an exon, a sequence encoding a transcription termination sequence, a sequence encoding a chloroplast targeting peptide, a sequence encoding a mitochondrial targeting peptide, an insulator sequence, a sequence encoding an anti-sense RNA construct, a sequence encoding non-protein-coding RNA (npcRNA), a sequence encoding a recombinase, a sequence encoding a recombinase recognition site, a landing pad, an editing template, an expression cassette, a stack of two or more expression cassettes encoding transgenes, a sequence encoding a site-specific enzyme, a sequence encoding a site-specific enzyme target site, a sequence encoding a selection marker, a sequence encoding a cell factor that functions to increase DNA repair
  • the sequence of interest comprises a sequence encoding a site-specific enzyme target site 5' to an expression cassette and a sequence encoding a site-specific enzyme target site 3' to an expression cassette.
  • the sequence of interest does not comprise a homology arm DNA sequence.
  • the sequence of interest comprises at least one homology arm DNA sequence.
  • the sequence of interest comprises both a left homology arm DNA sequence and a right homology arm DNA sequence.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, and ii) a second sequence that is not positioned between the left homology arm DNA sequence and the right homology arm DNA sequence.
  • the sequence of interest comprises a sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites. In some embodiments, the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence that is not positioned between sequences encoding site-specific enzyme target sites.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence encoding the site-specific enzyme, where the second sequence is not positioned between sequences encoding site-specific enzyme target sites.
  • at least one homology arm DNA sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a target sequence in the plant genome.
  • the target sequence in the plant genome is a genie sequence.
  • the target sequence in the plant genome is a non-genic sequence.
  • the sequence of interest flanked by homology arms comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a native gene of the plant cell.
  • the sequence of interest comprises a protein-coding sequence.
  • the sequence of interest comprises a non-protein-coding RNA.
  • the non- protein-coding RNA is selected from the group consisting of: a microRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), a small RNA (22-26 nt in length) and precursor encoding same, a heterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), a hairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA (ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a tracer RNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or any combination thereof.
  • miRNA microRNA
  • miRNA precursor a small interfering RNA
  • siRNA small interfering RNA
  • small RNA 22-26 nt in length
  • precursor precursor encoding same
  • hc-siRNA a heterochromatic siRNA
  • the site-specific enzyme is selected from a group consisting of an
  • the endonuclease is selected from a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasX nuclease, a CasY nuclease, and a Cpfl nuclease.
  • TALEN transcription activator-like effector nuclease
  • the Cas9 nuclease is selected from the group comprising Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4 nuclease.
  • the recombinase is a tyrosine
  • the tyrosine recombinase attached to a DNA recognition motif is selected from the group consisting of a Cre recombinase, a Flp recombinase, and a Tnpl recombinase.
  • the serine recombinase attached to a DNA recognition motif is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase.
  • the transposase is a DNA transposase attached to a DNA binding domain.
  • the sequence of interest further comprises at least one site-specific enzyme target site.
  • the at least one site-specific enzyme target site is selected from a Cre/lox recombination site, a Flp/FRT recombination site, a endonuclease recognition site, and a TALEN site.
  • the sequence of interest comprises a sequence encoding a protein involved in DNA repair, wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via homologous recombination, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via non-homologous end joining, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • the Rhizobiales cell is selected from an Agrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp., a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp.
  • the Rhizobiales cell further contains a vector comprising at least one expression cassette, wherein the expression cassettes comprise a sequence encoding a protein involved in DNA repair, and wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant- negative Ku70, or any combination thereof.
  • the plant cell already comprises a site-specific enzyme.
  • the plant cell is selected from the group consisting of a corn cell, a soybean cell, a canola cell, a cotton cell, a wheat cell, or a sugarcane cell.
  • a nucleotide sequence encoding the site-specific enzyme is stably transformed into the plant cell.
  • Several embodiments relate to a method of transforming a plant cell, comprising contacting the plant cell with two or more Rhizobiales cells capable of transforming the plant cell, wherein the two or more Rhizobiales cells each contain one of at least two vectors capable of forming two essentially complementary T-strands, wherein each vector comprises an essentially identical sequence of interest, and where the first vector comprises a RBI and a first left border DNA sequence (LBl) which are positioned in the first vector to initiate (RB I) and terminate (LB 1) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the second vector comprises a RB2 and a second left border DNA sequence (LB2) which are positioned in the second vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation from the 5' to 3' end of the second T-strand, and wherein the sequence of interest in the two T-
  • the RBI and the RB2 are essentially homologous. In some embodiments, the LBl and the LB2 are essentially homologous. In some embodiments, the RBI and the RB2 are not essentially homologous. In some embodiments, the LBl and LB2 are not essentially homologous. In some embodiments, at least one of the RBI and RB2 comprise an Agrobacterium Ti plasmid right border consensus DNA sequence. In some embodiments, the right border consensus DNA sequence is selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, at least one of the RBI and RB2 comprise a sequence selected from SEQ ID NOs: 1-13. In some
  • At least one of the RBI and RB2 comprise a sequence at least 80% identical to a sequence selected from SEQ ID NOs:4 and SEQ ID NO: 12.
  • at least one of the LB l and LB2 comprise an Agrobacterium Ti plasmid left border consensus DNA sequence.
  • the left border consensus DNA sequence is selected from SEQ ID NO: 23 or SEQ ID NO: 24.
  • at least one of the LBl and LB2 comprise a sequence selected from SEQ ID NOs: 14-20.
  • at least one of the LBl and LB2 comprise a sequence at least 80% identical to SEQ ID NO: 19.
  • the sequence of interest comprises one or more expression cassettes.
  • the sequence of interest comprises one or more sequences selected from: a gene, a portion of a gene, an intergenic sequence, an enhancer, a promoter, an intron, an exon, a sequence encoding a transcription termination sequence, a sequence encoding a chloroplast targeting peptide, a sequence encoding a mitochondrial targeting peptide, an insulator sequence, a sequence encoding an anti-sense RNA construct, a sequence encoding non-protein-coding RNA (npcRNA), a sequence encoding a recombinase, a sequence encoding a recombinase recognition site, a landing pad, an editing template, an expression cassette, a stack of two or more expression cassettes encoding transgenes, a sequence encoding a site-specific enzyme, a sequence encoding a site-specific enzyme target site, a sequence encoding a selection marker, a sequence encoding a cell factor that functions to increase DNA repair
  • the sequence of interest does not comprise a homology arm DNA sequence.
  • the sequence of interest comprises at least one homology arm DNA sequence.
  • the sequence of interest comprises both a left homology arm DNA sequence and a right homology arm DNA sequence.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, and ii) a second sequence that is not positioned between the left homology arm DNA sequence and the right homology arm DNA sequence.
  • the sequence of interest comprises a sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence that is not positioned between sequences encoding site-specific enzyme target sites.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence encoding the site-specific enzyme, where the second sequence is not positioned between sequences encoding site-specific enzyme target sites.
  • at least one homology arm DNA sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%), or at least 99% identical to a target sequence in the plant genome.
  • the target sequence in the plant genome is a genie sequence. In some embodiments, the target sequence in the plant genome is a non-genic sequence. In some embodiments, the sequence of interest flanked by homology arms comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least
  • the sequence of interest comprises a protein-coding sequence. In some embodiments, the sequence of interest comprises a non- protein-coding RNA.
  • the non-protein-coding RNA is selected from the group consisting of: a microRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), a small RNA (22-26 nt in length) and precursor encoding same, a heterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), a hairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA (ta-siRNA), a naturally occurring antisense siRNA (nat- siRNA), a tracer RNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or any combination thereof.
  • the site-specific enzyme is selected from a group consisting of an endonuclease, a recombinase, and a transposase.
  • the endonuclease is selected from a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasX nuclease, a CasY nuclease, and a Cpfl nuclease.
  • TALEN transcription activator-like effector nuclease
  • Argonaute a Cas9 nuclease
  • CasX nuclease a CasX nuclease
  • CasY nuclease a CasY nuclease
  • Cpfl nuclease a Cpfl nuclease
  • the Cas9 nuclease is selected from the group comprising Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4 nuclease.
  • the recombinase is a tyrosine recombinase attached to a DNA recognition motif, or a serine recombinase attached to a DNA recognition motif.
  • the tyrosine recombinase attached to a DNA recognition motif is selected from the group consisting of a Cre recombinase, a Flp recombinase, and a Tnpl recombinase.
  • the serine recombinase attached to a DNA recognition motif is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase.
  • the transposase is a DNA transposase attached to a DNA binding domain.
  • the sequence of interest further comprises at least one site-specific enzyme target site.
  • the at least one site-specific enzyme target site is selected from a Cre/lox recombination site, a Flp/FRT recombination site, a endonuclease recognition site, and a TALEN site.
  • the sequence of interest comprises a sequence encoding a protein involved in DNA repair, wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via homologous recombination, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via non-homologous end joining, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an
  • the plant cell already comprises a site-specific enzyme.
  • the plant cell is selected from the group consisting of a corn cell, a soybean cell, a canola cell, a cotton cell, a wheat cell, or a sugarcane cell.
  • a nucleotide sequence encoding the site-specific enzyme is stably transformed into the plant cell.
  • the Rhizobiales cell is selected from an Agrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp., a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp.
  • the Rhizobiales cell further contains a vector comprising at least one expression cassette, wherein the expression cassettes comprise a sequence encoding a protein involved in DNA repair, and wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • a method of increasing the rate of site directed integration of a sequence of interest comprising contacting a plant cell with at least one vector capable of forming two essentially complementary T-strands, wherein the at least one vector comprises a first right border DNA sequence (RBI), a second right border DNA sequence (RB2), and at least one sequence of interest, wherein the RBI is positioned in the vector to initiate synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the RB2 is positioned in the vector to initiate synthesis of a second T-strand such that the sequence of interest is in the anti-sense orientation relative to the sequence of interest in the first T-strand, and wherein the two T- strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the sequence of interest.
  • the at least one vector comprises a first right border DNA sequence (RBI), a second right border DNA sequence (RB2), and at least one sequence of interest
  • the RBI and the RB2 are essentially homologous. In some embodiments, the LBl and the LB2 are essentially homologous. In some embodiments, the RBI and the RB2 are not essentially homologous. In some embodiments, the LBl and LB2 are not essentially homologous. In some embodiments, at least one of the RBI and RB2 comprise an Agrobacterium Ti plasmid right border consensus DNA sequence. In some embodiments, the right border consensus DNA sequence is selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, at least one of the RBI and RB2 comprise a sequence selected from SEQ ID NOs: 1-13. In some
  • At least one of the RBI and RB2 comprise a sequence at least 80% identical to a sequence selected from SEQ ID NOs:4 and SEQ ID NO: 12.
  • at least one of the LB 1 and LB2 comprise an Agrobacterium Ti plasmid left border consensus DNA sequence.
  • the left border consensus DNA sequence is selected from SEQ ID NO: 23 or SEQ ID NO: 24.
  • at least one of the LB1 and LB2 comprise a sequence selected from SEQ ID NOs: 14-20.
  • at least one of the LB1 and LB2 comprise a sequence at least 80% identical to SEQ ID NO: 19.
  • the sequence of interest comprises one or more expression cassettes.
  • the sequence of interest comprises one or more sequences selected from: a gene, a portion of a gene, an intergenic sequence, an enhancer, a promoter, an intron, an exon, a sequence encoding a transcription termination sequence, a sequence encoding a chloroplast targeting peptide, a sequence encoding a mitochondrial targeting peptide, an insulator sequence, a sequence encoding an anti-sense RNA construct, a sequence encoding non-protein-coding RNA (npcRNA), a sequence encoding a recombinase, a sequence encoding a recombinase recognition site, a landing pad, an editing template, an expression cassette, a stack of two or more expression cassettes encoding transgenes, a sequence encoding a site-specific enzyme, a sequence encoding a site-specific enzyme target site, a sequence encoding a selection marker, a sequence encoding a cell factor that functions to increase DNA repair
  • the sequence of interest does not comprise a homology arm DNA sequence.
  • the sequence of interest comprises at least one homology arm DNA sequence.
  • the sequence of interest comprises both a left homology arm DNA sequence and a right homology arm DNA sequence.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, and ii) a second sequence that is not positioned between the left homology arm DNA sequence and the right homology arm DNA sequence.
  • the sequence of interest comprises a sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence that is not positioned between sequences encoding site-specific enzyme target sites.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence encoding the site-specific enzyme, where the second sequence is not positioned between sequences encoding site-specific enzyme target sites.
  • at least one homology arm DNA sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%), or at least 99% identical to a target sequence in the plant genome.
  • the target sequence in the plant genome is a genie sequence. In some embodiments, the target sequence in the plant genome is a non-genic sequence. In some embodiments, the sequence of interest flanked by homology arms comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a native gene of the plant cell. In some embodiments, the sequence of interest comprises a protein-coding sequence. In some embodiments, the sequence of interest comprises a non- protein-coding RNA.
  • the non-protein-coding RNA is selected from the group consisting of: a microRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), a small RNA (22-26 nt in length) and precursor encoding same, a heterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), a hairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA (ta-siRNA), a naturally occurring antisense siRNA (nat- siRNA), a tracer RNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or any combination thereof.
  • the site-specific enzyme is selected from a group consisting of an endonuclease, a recombinase, and a transposase.
  • the endonuclease is selected from a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasX nuclease, a CasY nuclease, and a Cpfl nuclease.
  • TALEN transcription activator-like effector nuclease
  • Argonaute a Cas9 nuclease
  • CasX nuclease a CasX nuclease
  • CasY nuclease a CasY nuclease
  • Cpfl nuclease a Cpfl nuclease
  • the Cas9 nuclease is selected from the group comprising Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4 nuclease.
  • the recombinase is a tyrosine recombinase attached to a DNA recognition motif, or a serine recombinase attached to a DNA recognition motif.
  • the tyrosine recombinase attached to a DNA recognition motif is selected from the group consisting of a Cre recombinase, a Flp recombinase, and a Tnpl recombinase.
  • the serine recombinase attached to a DNA recognition motif is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase.
  • the transposase is a DNA transposase attached to a DNA binding domain.
  • the sequence of interest further comprises at least one site-specific enzyme target site.
  • the at least one site-specific enzyme target site is selected from a Cre/lox recombination site, a Flp/FRT recombination site, a endonuclease recognition site, and a TALEN site.
  • the sequence of interest comprises a sequence encoding a protein involved in DNA repair, wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via homologous recombination, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via non-homologous end joining, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an
  • the plant cell already comprises a site-specific enzyme.
  • the plant cell is selected from the group consisting of a corn cell, a soybean cell, a canola cell, a cotton cell, a wheat cell, or a sugarcane cell.
  • a nucleotide sequence encoding the site-specific enzyme is stably transformed into the plant cell.
  • a method of increasing the rate of site directed integration of a sequence of interest comprising contacting a plant cell with at least one vector capable of forming two essentially complementary T-strands, wherein the at least one vector comprises a RBI, a RB2, a sequence of interest, a first left border DNA sequence (LBl), a second left border DNA sequence (LB2) and wherein the vector is configured such that the RBI is paired with the LBl which are positioned in the vector to initiate (RBI) and terminate (LBl) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the RB2 is paired with the LB2 which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation relative to the sequence of interest in the first T-strand, and wherein the sequence of interest in the two T- strands resulting from
  • the RBI and the RB2 are essentially homologous. In some embodiments, the LBl and the LB2 are essentially homologous. In some embodiments, the RBI and the RB2 are not essentially homologous. In some embodiments, the LBl and LB2 are not essentially homologous. In some embodiments, at least one of the RBI and RB2 comprise an Agrobacterium Ti plasmid right border consensus DNA sequence. In some embodiments, the right border consensus DNA sequence is selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, at least one of the RBI and RB2 comprise a sequence selected from SEQ ID NOs: 1-13. In some
  • At least one of the RBI and RB2 comprise a sequence at least 80% identical to a sequence selected from SEQ ID NOs:4 and SEQ ID NO: 12.
  • at least one of the LB l and LB2 comprise an Agrobacterium Ti plasmid left border consensus DNA sequence.
  • the left border consensus DNA sequence is selected from SEQ ID NO: 23 or SEQ ID NO: 24.
  • at least one of the LBl and LB2 comprise a sequence selected from SEQ ID NOs: 14-20.
  • at least one of the LBl and LB2 comprise a sequence at least 80% identical to SEQ ID NO: 19.
  • the sequence of interest comprises one or more expression cassettes.
  • the sequence of interest comprises one or more sequences selected from: a gene, a portion of a gene, an intergenic sequence, an enhancer, a promoter, an intron, an exon, a sequence encoding a transcription termination sequence, a sequence encoding a chloroplast targeting peptide, a sequence encoding a mitochondrial targeting peptide, an insulator sequence, a sequence encoding an anti-sense RNA construct, a sequence encoding non-protein-coding RNA (npcRNA), a sequence encoding a recombinase, a sequence encoding a recombinase recognition site, a landing pad, an editing template, an expression cassette, a stack of two or more expression cassettes encoding transgenes, a sequence encoding a site-specific enzyme, a sequence encoding a site-specific enzyme target site, a sequence encoding a selection marker, a sequence encoding a cell factor that functions to increase DNA repair
  • the sequence of interest does not comprise a homology arm DNA sequence.
  • the sequence of interest comprises at least one homology arm DNA sequence.
  • the sequence of interest comprises both a left homology arm DNA sequence and a right homology arm DNA sequence.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, and ii) a second sequence that is not positioned between the left homology arm DNA sequence and the right homology arm DNA sequence.
  • the sequence of interest comprises a sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence that is not positioned between sequences encoding site-specific enzyme target sites.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence encoding the site-specific enzyme, where the second sequence is not positioned between sequences encoding site-specific enzyme target sites.
  • at least one homology arm DNA sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%), or at least 99% identical to a target sequence in the plant genome.
  • the target sequence in the plant genome is a genie sequence. In some embodiments, the target sequence in the plant genome is a non-genic sequence. In some embodiments, the sequence of interest flanked by homology arms comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a native gene of the plant cell. In some embodiments, the sequence of interest comprises a protein-coding sequence. In some embodiments, the sequence of interest comprises a non- protein-coding RNA.
  • the non-protein-coding RNA is selected from the group consisting of: a microRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), a small RNA (22-26 nt in length) and precursor encoding same, a heterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), a hairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA (ta-siRNA), a naturally occurring antisense siRNA (nat- siRNA), a tracer RNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or any combination thereof.
  • the site-specific enzyme is selected from a group consisting of an endonuclease, a recombinase, and a transposase.
  • the endonuclease is selected from a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasX nuclease, a CasY nuclease, and a Cpfl nuclease.
  • TALEN transcription activator-like effector nuclease
  • Argonaute a Cas9 nuclease
  • CasX nuclease a CasX nuclease
  • CasY nuclease a CasY nuclease
  • Cpfl nuclease a Cpfl nuclease
  • the Cas9 nuclease is selected from the group comprising Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4 nuclease.
  • the recombinase is a tyrosine
  • the tyrosine recombinase attached to a DNA recognition motif is selected from the group consisting of a Cre recombinase, a Flp recombinase, and a Tnpl recombinase.
  • the serine recombinase attached to a DNA recognition motif is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase.
  • the transposase is a DNA transposase attached to a DNA binding domain.
  • the sequence of interest further comprises at least one site-specific enzyme target site.
  • the at least one site-specific enzyme target site is selected from a Cre/lox recombination site, a Flp/FRT recombination site, a endonuclease recognition site, and a TALEN site.
  • the sequence of interest comprises a sequence encoding a protein involved in DNA repair, wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via homologous recombination, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via non-homologous end joining, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an
  • the plant cell already comprises a site-specific enzyme.
  • the plant cell is selected from the group consisting of a corn cell, a soybean cell, a canola cell, a cotton cell, a wheat cell, or a sugarcane cell.
  • a nucleotide sequence encoding the site-specific enzyme is stably transformed into the plant cell.
  • Several embodiments relate to a method of increasing the rate of site directed integration of a sequence of interest, comprising contacting a plant cell with at least one vector capable of forming two essentially complementary T-strands, wherein the vector comprises a first sequence of interest and a second sequence of interest, wherein the first sequence of interest is essentially identical to the second sequence of interest; wherein the vector further comprises a RBI and a LBl which are positioned in the vector to initiate (RBI) and terminate (LB l) synthesis of a first T-strand such that the first sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the vector further comprises a RB2 and aLB2 which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the second sequence of interest is in an anti- sense orientation relative to the first sequence of interest in the first T-strand, and wherein the two T-strands resulting from initiation at RBI and RB2 are essentially complementary
  • the RBI and the RB2 are essentially homologous. In some embodiments, the LBl and the LB2 are essentially homologous. In some embodiments, the RBI and the RB2 are not essentially homologous. In some embodiments, the LBl and LB2 are not essentially homologous. In some embodiments, at least one of the RBI and RB2 comprise an Agrobacterium Ti plasmid right border consensus DNA sequence. In some embodiments, the right border consensus DNA sequence is selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, at least one of the RBI and RB2 comprise a sequence selected from SEQ ID NOs: 1-13.
  • At least one of the RBI and RB2 comprise a sequence at least 80% identical to a sequence selected from SEQ ID NOs:4 and SEQ ID NO: 12.
  • at least one of the LB l and LB2 comprise an Agrobacterium Ti plasmid left border consensus DNA sequence.
  • the left border consensus DNA sequence is selected from SEQ ID NO: 23 or SEQ ID NO: 24.
  • at least one of the LB l and LB2 comprise a sequence selected from SEQ ID NOs: 14-20.
  • at least one of the LBl and LB2 comprise a sequence at least 80% identical to SEQ ID NO: 19.
  • the sequence of interest comprises one or more expression cassettes.
  • the sequence of interest comprises one or more sequences selected from: a gene, a portion of a gene, an intergenic sequence, an enhancer, a promoter, an intron, an exon, a sequence encoding a transcription termination sequence, a sequence encoding a chloroplast targeting peptide, a sequence encoding a mitochondrial targeting peptide, an insulator sequence, a sequence encoding an anti-sense RNA construct, a sequence encoding non-protein-coding RNA (npcRNA), a sequence encoding a recombinase, a sequence encoding a recombinase recognition site, a landing pad, an editing template, an expression cassette, a stack of two or more expression cassettes encoding transgenes, a sequence encoding a site-specific enzyme, a sequence encoding a site-specific enzyme target site, a sequence encoding a selection marker, a sequence encoding a cell factor that functions to increase DNA repair
  • the sequence of interest comprises a sequence encoding a site-specific enzyme target site 5' to an expression cassette and a sequence encoding a site-specific enzyme target site 3' to an expression cassette.
  • the sequence of interest does not comprise a homology arm DNA sequence.
  • the sequence of interest comprises at least one homology arm DNA sequence.
  • the sequence of interest comprises both a left homology arm DNA sequence and a right homology arm DNA sequence.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, and ii) a second sequence that is not positioned between the left homology arm DNA sequence and the right homology arm DNA sequence.
  • the sequence of interest comprises a sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites. In some embodiments, the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence that is not positioned between sequences encoding site-specific enzyme target sites.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence encoding the site-specific enzyme, where the second sequence is not positioned between sequences encoding site-specific enzyme target sites.
  • at least one homology arm DNA sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a target sequence in the plant genome.
  • the target sequence in the plant genome is a genie sequence.
  • the target sequence in the plant genome is a non-genic sequence.
  • the sequence of interest flanked by homology arms comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), or 100% identical to a native gene of the plant cell.
  • the sequence of interest comprises a protein-coding sequence.
  • the sequence of interest comprises a non-protein-coding RNA.
  • the non- protein-coding RNA is selected from the group consisting of: a microRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), a small RNA (22-26 nt in length) and precursor encoding same, a heterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), a hairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA (ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a tracer RNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or any combination thereof.
  • miRNA microRNA
  • miRNA precursor a small interfering RNA
  • siRNA small interfering RNA
  • small RNA 22-26 nt in length
  • precursor precursor encoding same
  • hc-siRNA a heterochromatic siRNA
  • the site-specific enzyme is selected from a group consisting of an
  • the endonuclease is selected from a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasX nuclease, a CasY nuclease, and a Cpfl nuclease.
  • TALEN transcription activator-like effector nuclease
  • the Cas9 nuclease is selected from the group comprising Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csfi, and Csf4 nuclease.
  • the recombinase is a tyrosine
  • the tyrosine recombinase attached to a DNA recognition motif is selected from the group consisting of a Cre recombinase, a Flp recombinase, and a Tnpl recombinase.
  • the serine recombinase attached to a DNA recognition motif is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase.
  • the transposase is a DNA transposase attached to a DNA binding domain.
  • the sequence of interest further comprises at least one site-specific enzyme target site.
  • the at least one site-specific enzyme target site is selected from a Cre/lox recombination site, a Flp/FRT recombination site, a
  • the sequence of interest comprises a sequence encoding a protein involved in DNA repair, wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via homologous recombination, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via non-homologous end joining, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • the plant cell already comprises a site-specific enzyme.
  • the plant cell is selected from the group consisting of a corn cell, a soybean cell, a canola cell, a cotton cell, a wheat cell, or a sugarcane cell.
  • a nucleotide sequence encoding the site-specific enzyme is stably transformed into the plant cell.
  • a method of increasing the rate of site directed integration of a sequence of interest comprising contacting a plant cell with at least one vector capable of forming two essentially complementary T-strands, wherein the Rhizobiales cell comprises at least a first and a second vector, wherein each vector comprises essentially identical sequences of interest, and wherein the first vector comprises a RB 1 and a LB 1 which are positioned in the first vector to initiate (RB I) and terminate (LB 1) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the second vector comprises a RB2 and a LB2 which are positioned in the second vector to initiate (RB2) and terminate (LB2) synthesis of a second T- strand such that the sequence of interest is in an anti-sense orientation relative to the sequence of interest in the first T-strand, and wherein the two T-strands resulting from initiation at RBI and RB2 are essentially complementary in
  • the RBI and the RB2 are essentially homologous. In some embodiments, the LB1 and the LB2 are essentially homologous. In some embodiments, the RBI and the RB2 are not essentially homologous. In some embodiments, the LB1 and LB2 are not essentially homologous. In some embodiments, at least one of the RBI and RB2 comprise an Agrobacterium Ti plasmid right border consensus DNA sequence. In some embodiments, the right border consensus DNA sequence is selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, at least one of the RBI and RB2 comprise a sequence selected from SEQ ID NOs: 1-13.
  • At least one of the RBI and RB2 comprise a sequence at least 80% identical to a sequence selected from SEQ ID NOs:4 and SEQ ID NO: 12.
  • at least one of the LB 1 and LB2 comprise an Agrobacterium Ti plasmid left border consensus DNA sequence.
  • the left border consensus DNA sequence is selected from SEQ ID NO: 23 or SEQ ID NO: 24.
  • at least one of the LB 1 and LB2 comprise a sequence selected from SEQ ID NOs: 14-20.
  • at least one of the LB1 and LB2 comprise a sequence at least 80% identical to SEQ ID NO: 19.
  • the sequence of interest comprises one or more expression cassettes.
  • the sequence of interest comprises one or more sequences selected from: a gene, a portion of a gene, an intergenic sequence, an enhancer, a promoter, an intron, an exon, a sequence encoding a transcription termination sequence, a sequence encoding a chloroplast targeting peptide, a sequence encoding a mitochondrial targeting peptide, an insulator sequence, a sequence encoding an anti-sense RNA construct, a sequence encoding non-protein-coding RNA (npcRNA), a sequence encoding a recombinase, a sequence encoding a recombinase recognition site, a landing pad, an editing template, an expression cassette, a stack of two or more expression cassettes encoding transgenes, a sequence encoding a site-specific enzyme, a sequence encoding a site-specific enzyme target site, a sequence encoding a selection marker, a sequence encoding a cell factor that functions to increase DNA repair
  • the sequence of interest comprises a sequence encoding a site-specific enzyme target site 5' to an expression cassette and a sequence encoding a site-specific enzyme target site 3' to an expression cassette.
  • the sequence of interest does not comprise a homology arm DNA sequence.
  • the sequence of interest comprises at least one homology arm DNA sequence.
  • the sequence of interest comprises both a left homology arm DNA sequence and a right homology arm DNA sequence.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, and ii) a second sequence that is not positioned between the left homology arm DNA sequence and the right homology arm DNA sequence.
  • the sequence of interest comprises a sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites. In some embodiments, the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence that is not positioned between sequences encoding site-specific enzyme target sites.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence encoding the site-specific enzyme, where the second sequence is not positioned between sequences encoding site-specific enzyme target sites.
  • at least one homology arm DNA sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a target sequence in the plant genome.
  • the target sequence in the plant genome is a genie sequence.
  • the target sequence in the plant genome is a non-genic sequence.
  • the sequence of interest flanked by homology arms comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), or 100% identical to a native gene of the plant cell.
  • the sequence of interest comprises a protein-coding sequence.
  • the sequence of interest comprises a non-protein-coding RNA.
  • the non- protein-coding RNA is selected from the group consisting of: a microRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), a small RNA (22-26 nt in length) and precursor encoding same, a heterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), a hairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA (ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a tracer RNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or any combination thereof.
  • miRNA microRNA
  • miRNA precursor a small interfering RNA
  • siRNA small interfering RNA
  • small RNA 22-26 nt in length
  • precursor precursor encoding same
  • hc-siRNA a heterochromatic siRNA
  • the site-specific enzyme is selected from a group consisting of an
  • the endonuclease is selected from a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasX nuclease, a CasY nuclease, and a Cpfl nuclease.
  • TALEN transcription activator-like effector nuclease
  • the Cas9 nuclease is selected from the group comprising Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4 nuclease.
  • the recombinase is a tyrosine
  • the tyrosine recombinase attached to a DNA recognition motif is selected from the group consisting of a Cre recombinase, a Flp recombinase, and a Tnpl recombinase.
  • the serine recombinase attached to a DNA recognition motif is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase.
  • the transposase is a DNA transposase attached to a DNA binding domain.
  • the sequence of interest further comprises at least one site-specific enzyme target site.
  • the at least one site-specific enzyme target site is selected from a Cre/lox recombination site, a Flp/FRT recombination site, a endonuclease recognition site, and a TALEN site.
  • the sequence of interest comprises a sequence encoding a protein involved in DNA repair, wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via homologous recombination, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via non-homologous end joining, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • the Rhizobiales cell is selected from an Agrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp., a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp.
  • the Rhizobiales cell further contains a vector comprising at least one expression cassette, wherein the expression cassettes comprise a sequence encoding a protein involved in DNA repair, and wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant- negative Ku70, or any combination thereof.
  • the plant cell already comprises a site-specific enzyme.
  • the plant cell is selected from the group consisting of a corn cell, a soybean cell, a canola cell, a cotton cell, a wheat cell, or a sugarcane cell.
  • a nucleotide sequence encoding the site-specific enzyme is stably transformed into the plant cell.
  • a method of increasing the rate of site directed integration of a sequence of interest comprising contacting a plant cell with at least one vector capable of forming two essentially complementary T-strands, wherein the at least one vector comprises a first sequence of interest, a second sequence of interest different from the first sequence of interest, at least two RB DNA sequences, and one or more optional LB DNA sequences, wherein the first RB DNA sequence (RBI) and a first LB DNA sequence (LBl) are positioned in the vector to initiate (RBI) and terminate (LBl) synthesis of the first T-strand such that the first sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the vector configuration further comprises a second RB DNA sequence (RB2) and a second LB DNA sequence (LB2) which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the first sequence of interest is in an anti-sense orientation from the 5' to 3'
  • the RBI and the RB2 are essentially homologous. In some embodiments, the LBl and the LB2 are essentially homologous. In some embodiments, the RBI and the RB2 are not essentially homologous. In some embodiments, the LBl and LB2 are not essentially homologous. In some embodiments, at least one of the RBI and RB2 comprise an Agrobacterium Ti plasmid right border consensus DNA sequence. In some embodiments, the right border consensus DNA sequence is selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, at least one of the RBI and RB2 comprise a sequence selected from SEQ ID NOs: 1-13.
  • At least one of the RBI and RB2 comprise a sequence at least 80% identical to a sequence selected from SEQ ID NOs:4 and SEQ ID NO: 12.
  • at least one of the LBl and LB2 comprise an Agrobacterium Ti plasmid left border consensus DNA sequence.
  • the left border consensus DNA sequence is selected from SEQ ID NO: 23 or SEQ ID NO: 24.
  • at least one of the LBl and LB2 comprise a sequence selected from SEQ ID NOs: 14-20.
  • at least one of the LBl and LB2 comprise a sequence at least 80% identical to SEQ ID NO: 19.
  • the sequence of interest comprises one or more expression cassettes.
  • the sequence of interest comprises one or more sequences selected from: a gene, a portion of a gene, an intergenic sequence, an enhancer, a promoter, an intron, an exon, a sequence encoding a transcription termination sequence, a sequence encoding a chloroplast targeting peptide, a sequence encoding a mitochondrial targeting peptide, an insulator sequence, a sequence encoding an anti-sense RNA construct, a sequence encoding non-protein-coding RNA (npcRNA), a sequence encoding a recombinase, a sequence encoding a recombinase recognition site, a landing pad, an editing template, an expression cassette, a stack of two or more expression cassettes encoding transgenes, a sequence encoding a site-specific enzyme, a sequence encoding a site-specific enzyme target site, a sequence encoding a selection marker, a sequence encoding a cell factor that functions to increase DNA repair
  • the sequence of interest comprises a sequence encoding a site-specific enzyme target site 5' to an expression cassette and a sequence encoding a site-specific enzyme target site 3' to an expression cassette.
  • the sequence of interest does not comprise a homology arm DNA sequence.
  • the sequence of interest comprises at least one homology arm DNA sequence.
  • the sequence of interest comprises both a left homology arm DNA sequence and a right homology arm DNA sequence.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, and ii) a second sequence that is not positioned between the left homology arm DNA sequence and the right homology arm DNA sequence.
  • the sequence of interest comprises a sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites. In some embodiments, the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence that is not positioned between sequences encoding site-specific enzyme target sites.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence encoding the site-specific enzyme, where the second sequence is not positioned between sequences encoding site-specific enzyme target sites.
  • at least one homology arm DNA sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a target sequence in the plant genome.
  • the target sequence in the plant genome is a genie sequence.
  • the target sequence in the plant genome is a non-genic sequence.
  • the sequence of interest flanked by homology arms comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), or 100% identical to a native gene of the plant cell.
  • the sequence of interest comprises a protein-coding sequence.
  • the sequence of interest comprises a non-protein-coding RNA.
  • the non- protein-coding RNA is selected from the group consisting of: a microRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), a small RNA (22-26 nt in length) and precursor encoding same, a heterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), a hairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA (ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a tracer RNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or any combination thereof.
  • miRNA microRNA
  • miRNA precursor a small interfering RNA
  • siRNA small interfering RNA
  • small RNA 22-26 nt in length
  • precursor precursor encoding same
  • hc-siRNA a heterochromatic siRNA
  • the site-specific enzyme is selected from a group consisting of an
  • the endonuclease is selected from a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasX nuclease, a CasY nuclease, and a Cpfl nuclease.
  • TALEN transcription activator-like effector nuclease
  • the Cas9 nuclease is selected from the group comprising Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4 nuclease.
  • the recombinase is a tyrosine
  • the tyrosine recombinase attached to a DNA recognition motif is selected from the group consisting of a Cre recombinase, a Flp recombinase, and a Tnpl recombinase.
  • the serine recombinase attached to a DNA recognition motif is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase.
  • the transposase is a DNA transposase attached to a DNA binding domain.
  • the sequence of interest further comprises at least one site-specific enzyme target site.
  • the at least one site-specific enzyme target site is selected from a Cre/lox recombination site, a Flp/FRT recombination site, a
  • the sequence of interest comprises a sequence encoding a protein involved in DNA repair, wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via homologous recombination, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via non-homologous end joining, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • the plant cell already comprises a site-specific enzyme.
  • the plant cell is selected from the group consisting of a corn cell, a soybean cell, a canola cell, a cotton cell, a wheat cell, or a sugarcane cell.
  • a nucleotide sequence encoding the site-specific enzyme is stably transformed into the plant cell.
  • a method of transforming a plant cell comprising contacting the plant cell with a Rhizobiales cell capable of transforming the plant cell, wherein the Rhizobiales cell comprises at least one vector comprising a right border DNA sequence (RB) and a left border DNA sequence (LB) and wherein the vector comprises between the RB and LB: (i) a first sequence of interest in a sense orientation relative to the RB, (ii) a spacer, and (iii) a second sequence of interest in an anti-sense orientation relative to the RB, wherein the first sequence of interest and second sequence of interest are essentially complementary and after synthesis of the T-strand anneal to form a double-stranded DNA.
  • RB right border DNA sequence
  • LB left border DNA sequence
  • the first sequence of interest further comprises a first left homology arm DNA sequence and a first right homology arm DNA sequence
  • the second sequence of interest further comprises a second left homology arm DNA sequence and a second right homology arm DNA sequence.
  • the Rhizobiales cell is selected from an Agrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp., a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp.
  • the Rhizobiales cell further contains a vector comprising at least one expression cassette, wherein the expression cassettes comprise a sequence encoding a protein involved in DNA repair, and wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • the RBI and the RB2 are essentially
  • the LBl and the LB2 are essentially homologous. In some embodiments, the RBI and the RB2 are not essentially homologous. In some embodiments, the LBl and LB2 are not essentially homologous. In some embodiments, at least one of the RBI and RB2 comprise an Agrobacterium Ti plasmid right border consensus DNA sequence. In some embodiments, the right border consensus DNA sequence is selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, at least one of the RBI and RB2 comprise a sequence selected from SEQ ID NOs: 1-13.
  • At least one of the RBI and RB2 comprise a sequence at least 80% identical to a sequence selected from SEQ ID NOs:4 and SEQ ID NO: 12.
  • at least one of the LB l and LB2 comprise an Agrobacterium Ti plasmid left border consensus DNA sequence.
  • the left border consensus DNA sequence is selected from SEQ ID NO: 23 or SEQ ID NO: 24.
  • at least one of the LBl and LB2 comprise a sequence selected from SEQ ID NOs: 14-20.
  • at least one of the LBl and LB2 comprise a sequence at least 80% identical to SEQ ID NO: 19.
  • the sequence of interest comprises one or more expression cassettes.
  • the sequence of interest comprises one or more sequences selected from: a gene, a portion of a gene, an intergenic sequence, an enhancer, a promoter, an intron, an exon, a sequence encoding a transcription termination sequence, a sequence encoding a chloroplast targeting peptide, a sequence encoding a mitochondrial targeting peptide, an insulator sequence, a sequence encoding an anti-sense RNA construct, a sequence encoding non-protein-coding RNA (npcRNA), a sequence encoding a recombinase, a sequence encoding a recombinase recognition site, a landing pad, an editing template, an expression cassette, a stack of two or more expression cassettes encoding transgenes, a sequence encoding a site-specific enzyme, a sequence encoding a site-specific enzyme target site, a sequence encoding a selection marker, a sequence encoding a cell factor that functions to increase DNA repair
  • the sequence of interest comprises a sequence encoding a site-specific enzyme target site 5' to an expression cassette and a sequence encoding a site-specific enzyme target site 3' to an expression cassette.
  • the sequence of interest does not comprise a homology arm DNA sequence.
  • the sequence of interest comprises at least one homology arm DNA sequence.
  • the sequence of interest comprises both a left homology arm DNA sequence and a right homology arm DNA sequence.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, and ii) a second sequence that is not positioned between the left homology arm DNA sequence and the right homology arm DNA sequence.
  • the sequence of interest comprises a sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites. In some embodiments, the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence that is not positioned between sequences encoding site-specific enzyme target sites.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence encoding the site-specific enzyme, where the second sequence is not positioned between sequences encoding site-specific enzyme target sites.
  • at least one homology arm DNA sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a target sequence in the plant genome.
  • the target sequence in the plant genome is a genie sequence.
  • the target sequence in the plant genome is a non-genic sequence.
  • the sequence of interest flanked by homology arms comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), or 100% identical to a native gene of the plant cell.
  • the sequence of interest comprises a protein-coding sequence.
  • the sequence of interest comprises a non-protein-coding RNA.
  • the non- protein-coding RNA is selected from the group consisting of: a microRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), a small RNA (22-26 nt in length) and precursor encoding same, a heterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), a hairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA (ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a tracer RNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or any combination thereof.
  • miRNA microRNA
  • miRNA precursor a small interfering RNA
  • siRNA small interfering RNA
  • small RNA 22-26 nt in length
  • precursor precursor encoding same
  • hc-siRNA a heterochromatic siRNA
  • the site-specific enzyme is selected from a group consisting of an
  • the endonuclease is selected from a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasX nuclease, a CasY nuclease, and a Cpfl nuclease.
  • TALEN transcription activator-like effector nuclease
  • the Cas9 nuclease is selected from the group comprising Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4 nuclease.
  • the recombinase is a tyrosine
  • the tyrosine recombinase attached to a DNA recognition motif is selected from the group consisting of a Cre recombinase, a Flp recombinase, and a Tnpl recombinase.
  • the serine recombinase attached to a DNA recognition motif is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase.
  • the transposase is a DNA transposase attached to a DNA binding domain.
  • the sequence of interest further comprises at least one site-specific enzyme target site.
  • the at least one site-specific enzyme target site is selected from a Cre/lox recombination site, a Flp/FRT recombination site, a endonuclease recognition site, and a TALEN site.
  • the sequence of interest comprises a sequence encoding a protein involved in DNA repair, wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via homologous recombination, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via non-homologous end joining, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • the Rhizobiales cell is selected from an Agrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp., a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp.
  • the Rhizobiales cell further contains a vector comprising at least one expression cassette, wherein the expression cassettes comprise a sequence encoding a protein involved in DNA repair, and wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant- negative Ku70, or any combination thereof.
  • the plant cell already comprises a site-specific enzyme.
  • the plant cell is selected from the group consisting of a corn cell, a soybean cell, a canola cell, a cotton cell, a wheat cell, or a sugarcane cell.
  • a nucleotide sequence encoding the site-specific enzyme is stably transformed into the plant cell.
  • RB right border DNA sequence
  • LB left border DNA sequence
  • the vector comprises between the RB and LB: (i) a first sequence of interest in a sense orientation relative to the RB, (ii) a spacer, and (iii) a second sequence of interest in an anti-sense orientation relative to the RB, wherein the first sequence of interest and second sequence of interest are essentially complementary and after synthesis of the T-strand anneal to form a double-stranded DNA.
  • the first sequence of interest further comprises a first left homology arm DNA sequence and a first right homology arm DNA sequence
  • the second sequence of interest further comprises a second left homology arm DNA sequence and a second right homology arm
  • the RBI and the RB2 are essentially homologous. In some embodiments, the LBl and the LB2 are essentially homologous. In some embodiments, the RBI and the RB2 are not essentially homologous. In some embodiments, the LBl and LB2 are not essentially homologous. In some embodiments, at least one of the RBI and RB2 comprise an Agrobacterium Ti plasmid right border consensus DNA sequence. In some embodiments, the right border consensus DNA sequence is selected from SEQ ID NO: 21 or SEQ ID NO: 22. In some embodiments, at least one of the RBI and RB2 comprise a sequence selected from SEQ ID NOs: 1-13.
  • At least one of the RBI and RB2 comprise a sequence at least 80% identical to a sequence selected from SEQ ID NOs:4 and SEQ ID NO: 12.
  • at least one of the LBl and LB2 comprise an Agrobacterium Ti plasmid left border consensus DNA sequence.
  • the left border consensus DNA sequence is selected from SEQ ID NO: 23 or SEQ ID NO: 24.
  • at least one of the LBl and LB2 comprise a sequence selected from SEQ ID NOs: 14-20.
  • at least one of the LBl and LB2 comprise a sequence at least 80% identical to SEQ ID NO: 19.
  • the sequence of interest comprises one or more expression cassettes.
  • the sequence of interest comprises one or more sequences selected from: a gene, a portion of a gene, an intergenic sequence, an enhancer, a promoter, an intron, an exon, a sequence encoding a transcription termination sequence, a sequence encoding a chloroplast targeting peptide, a sequence encoding a mitochondrial targeting peptide, an insulator sequence, a sequence encoding an anti-sense RNA construct, a sequence encoding non-protein-coding RNA (npcRNA), a sequence encoding a recombinase, a sequence encoding a recombinase recognition site, a landing pad, an editing template, an expression cassette, a stack of two or more expression cassettes encoding transgenes, a sequence encoding a site-specific enzyme, a sequence encoding a site-specific enzyme target site, a sequence encoding a selection marker, a sequence encoding a cell factor that functions to increase DNA repair
  • the sequence of interest comprises a sequence encoding a site-specific enzyme target site 5' to an expression cassette and a sequence encoding a site-specific enzyme target site 3' to an expression cassette.
  • the sequence of interest does not comprise a homology arm DNA sequence.
  • the sequence of interest comprises at least one homology arm DNA sequence.
  • the sequence of interest comprises both a left homology arm DNA sequence and a right homology arm DNA sequence.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, and ii) a second sequence that is not positioned between the left homology arm DNA sequence and the right homology arm DNA sequence.
  • the sequence of interest comprises a sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence, where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites. In some embodiments, the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence that is not positioned between sequences encoding site-specific enzyme target sites.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology DNA arm sequence where the left and right homology arm sequences are positioned between two sequences encoding site-specific enzyme target sites, and ii) a second sequence encoding the site-specific enzyme, where the second sequence is not positioned between sequences encoding site-specific enzyme target sites.
  • at least one homology arm DNA sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a target sequence in the plant genome.
  • the target sequence in the plant genome is a genie sequence.
  • the target sequence in the plant genome is a non-genic sequence.
  • the sequence of interest flanked by homology arms comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), or 100% identical to a native gene of the plant cell.
  • the sequence of interest comprises a protein-coding sequence.
  • the sequence of interest comprises a non-protein-coding RNA.
  • the non- protein-coding RNA is selected from the group consisting of: a microRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), a small RNA (22-26 nt in length) and precursor encoding same, a heterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), a hairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA (ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a tracer RNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or any combination thereof.
  • miRNA microRNA
  • miRNA precursor a small interfering RNA
  • siRNA small interfering RNA
  • small RNA 22-26 nt in length
  • precursor precursor encoding same
  • hc-siRNA a heterochromatic siRNA
  • the site-specific enzyme is selected from a group consisting of an
  • the endonuclease is selected from a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasX nuclease, a CasY nuclease, and a Cpfl nuclease.
  • TALEN transcription activator-like effector nuclease
  • the Cas9 nuclease is selected from the group comprising Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4 nuclease.
  • the recombinase is a tyrosine
  • the tyrosine recombinase attached to a DNA recognition motif is selected from the group consisting of a Cre recombinase, a Flp recombinase, and a Tnpl recombinase.
  • the serine recombinase attached to a DNA recognition motif is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase.
  • the transposase is a DNA transposase attached to a DNA binding domain.
  • the sequence of interest further comprises at least one site-specific enzyme target site.
  • the at least one site-specific enzyme target site is selected from a Cre/lox recombination site, a Flp/FRT recombination site, a
  • the sequence of interest comprises a sequence encoding a protein involved in DNA repair, wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via homologous recombination, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • At least part of the sequence of interest is integrated into the plant genome via non-homologous end joining, wherein the integration of at least part of the sequence of interest results in a point mutation, an insertion, a deletion, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus, altered protein activity, altered RNAi products, altered RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • the plant cell already comprises a site-specific enzyme.
  • the plant cell is selected from the group consisting of a corn cell, a soybean cell, a canola cell, a cotton cell, a wheat cell, or a sugarcane cell.
  • a nucleotide sequence encoding the site-specific enzyme is stably transformed into the plant cell.
  • the Agrobacterium cell comprises at least one vector that is capable of forming two essentially complementary T-strands.
  • the Agrobacterium cell comprises at least one vector comprises a first right border DNA sequence (RBI), a second right border DNA sequence (RB2), and at least one sequence of interest, and wherein the RBI is positioned in the vector to initiate synthesis of a first T- strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the RB2 is positioned in the vector to initiate synthesis of a second T- strand such that the sequence of interest is in the anti-sense orientation relative to the sequence of interest in the first T-strand, and wherein the two T-strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the sequence of interest.
  • RBI right border DNA sequence
  • RB2 second right border DNA sequence
  • the Agrobacterium cell comprises at least one vector comprises a first right border DNA sequence (RBI), a second right border DNA sequence (RB2), a sequence of interest, a first left border DNA sequence (LBl) and a second left border DNA sequence (LB2), wherein the vector is configured such that the RBI is paired with the LBl which are positioned in the vector to initiate (RBI) and terminate (LB l) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the RB2 is paired with the LB2 which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation relative to the sequence of interest in the first T-strand, and wherein the two T-strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the sequence of interest.
  • RBI right border DNA sequence
  • RB2 second right border
  • the Agrobacterium cell comprises at least one vector comprises a first sequence of interest and a second sequence of interest, where the first sequence of interest is essentially identical to the second sequence of interest; wherein the vector further comprises a first right border DNA sequence (RBI) and a first left border DNA sequence (LBl) which are positioned in the vector to initiate (RBI) and terminate (LBl) synthesis of a first T-strand such that the first sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the vector further comprises a second right border DNA sequence (RB2) and a second left border DNA sequence (LB2) which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the second sequence of interest is in an anti-sense orientation relative to the first sequence of interest in the first T- strand, and wherein the two T-strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the first sequence of interest and the second sequence of
  • the Agrobacterium cell comprises at least a first vector and a second vector wherein each vector comprises essentially identical sequences of interest, and wherein the first vector comprises a first right border DNA sequence (RBI) and a first left border DNA sequence (LB 1) which are positioned in the first vector to initiate (RB 1) and terminate (LB 1) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and wherein the second vector comprises a second right border DNA sequence (RB2) and a second left border DNA sequence (LB2) which are positioned in the second vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation relative to the sequence of interest in the first T-strand, and wherein the two T- strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the sequence of interest.
  • the first vector comprises a first right border DNA sequence (RBI) and a first left
  • the Agrobacterium cell comprises a RBI and RB2 that are essentially homologous. In some embodiments, the Agrobacterium cell comprises a LB 1 and LB2that are essentially homologous. In some embodiments, the Agrobacterium cell comprises RBI and RB2 sequences that are not essentially homologous. In some embodiments, the Agrobacterium cell of LB 1 and LB2 sequences that are not essentially homologous. In some embodiments, at least one of the RBI and RB2 comprise an Agrobacterium Ti plasmid right border consensus DNA sequence. In some embodiments, the right border consensus DNA sequence is selected from SEQ ID NO: 21 or SEQ ID NO: 22.
  • At least one of the RBI and RB2 comprise a sequence selected from SEQ ID NOs: 1-13. In some embodiments, at least one of the RBI and RB2 comprise a sequence at least 80% identical to a sequence selected from SEQ ID NOs:4 and SEQ ID NO: 12. In some embodiments, at least one of the LB 1 and LB2 comprise an Agrobacterium Ti plasmid left border consensus DNA sequence. In some embodiments, the left border consensus DNA sequence is selected from SEQ ID NO: 23 or SEQ ID NO: 24 In some embodiments, at least one of the LB 1 and LB2 comprise a sequence selected from SEQ ID NOs: 14-20.
  • the sequence of interest comprises one or more expression cassettes.
  • the sequence of interest comprises at least one sequence selected from: a gene, a portion of a gene, an intergenic sequence, an enhancer, a promoter, an intron, an exon, a sequence encoding a transcription termination sequence, a sequence encoding a chloroplast targeting peptide, a sequence encoding a mitochondrial targeting peptide, an insulator sequence, a sequence encoding an anti-sense RNA construct, a sequence encoding non-protein-coding RNA (npcRNA), a sequence encoding a recombinase, a sequence encoding a recombinase recognition site, a landing pad, an editing template, an expression cassette, a stack of two or more expression cassettes encoding transgenes, a sequence encoding
  • sequence of interest does not comprise a homology arm DNA sequence. In some embodiments, the sequence of interest further comprises at least one homology arm DNA sequence. In some embodiments, the sequence of interest comprises both a left homology arm DNA sequence and a right homology arm DNA sequence.
  • the sequence of interest comprises i) a first sequence positioned between the left homology arm DNA sequence and the right homology arm DNA sequence, and ii) a second sequence that is not positioned between the region comprising the left homology arm DNA sequence and the right homology arm DNA sequence
  • the at least one homology arm DNA sequence comprises a sequence that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to a target sequence in the plant genome.
  • the sequence of interest comprises a protein-coding sequence.
  • the sequence of interest comprises a non-protein-coding RNA
  • the non-protein-coding RNA is selected from a microRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), a small RNA (22-26 nt in length) and precursor encoding same, a heterochromatic siRNA (hc-siRNA), a Piwi- interacting RNA (piRNA), a hairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA (ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a tracer RNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA), or any combination thereof.
  • the expression cassette comprises a nucleic acid sequence at least 80%) identical to a native plant gene. In some embodiments, the expression cassette comprises a nucleic acid sequence that is not homologous to a native plant sequence. In some embodiments, at least one expression cassette comprises at a sequence selected from: an insecticidal resistance gene, herbicide tolerance gene, nitrogen use efficiency gene, a water use efficiency gene, a nutritional quality gene, a DNA binding gene, a selectable marker gene, an RNAi construct, a site specific nuclease gene, a guide RNA, and any combination thereof.
  • the site-specific enzyme is selected from an endonuclease, a recombinase, a transposase, and any combination thereof.
  • the endonuclease is selected from the group consisting of: a meganuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute, a Cas9 nuclease, a CasX nuclease, a CasY nuclease, and a Cpfl nuclease.
  • the Cas9 nuclease is selected from the group comprising Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, and Csf4 nuclease.
  • the recombinase is a tyrosine recombinase attached to a DNA recognition motif, or a serine recombinase attached to a DNA recognition motif.
  • the tyrosine recombinase attached to a DNA recognition motif is selected from the group consisting of a Cre recombinase, a Flp recombinase, and a Tnpl recombinase.
  • the serine recombinase attached to a DNA recognition motif is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase.
  • the transposase is a DNA transposase attached to a DNA binding domain.
  • the sequence of interest comprises at least one site-specific enzyme target site.
  • at least one site-specific enzyme target site is selected from the group consisting of: a Cre/lox recombination site, a Flp/FRT recombination site, a endonuclease recognition site, and a
  • the sequence of interest comprises a sequence encoding at least one protein involved in DNA repair, wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • an Agrobacterium cell comprising at least one vector comprising a right border DNA sequence (RB) and a left border DNA sequence (LB) and wherein the vector comprises between the RB and LB: (i) a first sequence of interest in a sense orientation relative to the RB, (ii) a spacer, and (iii) a second sequence of interest in an anti-sense orientation relative to the RB, wherein the first sequence of interest and second sequence of interest are essentially complementary, and after synthesis of the T-strand anneal to form a double-stranded DNA.
  • RB right border DNA sequence
  • LB left border DNA sequence
  • the Agrobacterium cell comprises a first sequence of interest comprising a left homology arm DNA sequence and a right homology arm DNA sequence and a second sequence of interest comprising a left homology arm sequence DNA sequence and a right homology arm DNA sequence.
  • the RB comprises an Agrobacterium Ti plasmid right border consensus DNA sequence.
  • the right border consensus DNA sequence is selected from SEQ ID NO: 21 or SEQ ID NO: 22.
  • the RB comprises a sequence selected from SEQ ID NOs: 1-13.
  • the RB comprises a sequence at least 80% identical to a sequence selected from SEQ ID NOs:4 and SEQ ID NO: 12.
  • the LB comprises an Agrobacterium Ti plasmid left border consensus DNA sequence.
  • the left border consensus DNA sequence is selected from SEQ ID NO: 23 or SEQ ID NO: 24.
  • the LB comprises a sequence selected from SEQ ID NOs: 14-20.
  • the LB comprises a sequence at least 80%) identical to SEQ ID NO: 19.
  • a sequence of interest comprises one or more expression cassettes.
  • a sequence of interest comprises at least one sequence selected from: a gene, a portion of a gene, an intergenic sequence, an enhancer, a promoter, an intron, an exon, a sequence encoding a transcription termination sequence, a sequence encoding a chloroplast targeting peptide, a sequence encoding a mitochondrial targeting peptide, an insulator sequence, a sequence encoding an anti-sense RNA construct, a sequence encoding non-protein-coding RNA (npcRNA), a sequence encoding a recombinase, a sequence encoding a recombinase recognition site, a landing pad, an editing template, an expression cassette, a stack of two or more expression cassettes encoding transgenes, a sequence encoding a site-specific enzyme, a sequence encoding a site-specific enzyme target site, a sequence encoding a selection marker, gene expression cassette comprising a sequence encoding a cell factor that
  • Several embodiments relate to a method of transforming a plant genome, comprising contacting at least one plant cell on a co-culture medium for at least 24-48 hours, for at least 24-30 hours, for at least 30-36 hours, for at least 36-42 hours, for at least 42-48 hours, for at least 48-54 hours, for at least 54-60 hours, for at least 60-66 hours, for at least 66-72 hours, for at least 72-78 hours, for at least 78-84 hours, for at least 84-90 hours, for at least 90-96 hours, for at least 96-102 hours, for at least 102-108 hours, for at least 108-114 hours, for at least 114-120 hours, for at least 120-126 hours, or for at least 126-132 hours, with at least one Rhizobiales cell capable of transforming the plant cell,
  • Rhizobiales cell comprises at least one vector capable of forming two essentially
  • the plant cell is a corn immature embryo cell, a corn mature embryo cell, a corn seed cell, a soybean immature embryo cell, a soybean mature embryo cell, a soybean seed cell, a canola immature embryo cell, a canola mature embryo cell, a canola seed cell, a cotton immature embryo cell, a cotton mature embryo cell, a cotton seed cell, a wheat immature embryo cell, a wheat mature embryo cell, a wheat seed cell, a sugarcane immature embryo cell, a sugarcane mature embryo cell, or a sugarcane seed cell.
  • the Rhizobiales cell comprises at least one vector comprises a first right border (RBI) DNA sequence, a second right border DNA sequence (RB2), and at least one sequence of interest
  • the RBI is positioned in the vector to initiate synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand
  • the RB2 is positioned in the vector to initiate synthesis of a second T-strand such that the sequence of interest is in the anti-sense orientation relative to the sequence of interest in the first T-strand
  • the sequence of interest in the two T-strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the sequence of interest.
  • the Rhizobiales cell comprises at least one vector disclosed herein comprises a RBI, a RB2 , and a sequence of interest, and further comprises a first left border DNA sequence (LB 1) and a second left border DNA sequence (LB2), and wherein the vector is configured such that the RBI is paired with the LBl which are positioned in the vector to initiate (RBI) and terminate (LBl) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the RB2 is paired with the LB2 which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation relative to the sequence of interest in the first T-strand, and wherein the sequence of interest in the two T-strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the sequence of interest.
  • the vector is configured such that the RBI is paired with the
  • the Rhizobiales cell comprises a vector comprising a first sequence of interest and a second sequence of interest, where the first sequence of interest is essentially identical to the second sequence of interest; and the vector configuration further comprises a RB I with aLB l which are positioned in the vector to initiate (RBI) and terminate (LB l) synthesis of a first T-strand such that the first sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the vector further comprises a RB2 and a LB2 which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the second sequence of interest is in an anti-sense orientation relative to the first sequence of interest in the first T-strand, and wherein the sequence of interest in the two T-strands resulting from initiation at RB 1 and RB2 are essentially complementary in at least a portion of the first sequence of interest and the second sequence of interest.
  • the Rhizobiales cell comprises at least a first vector and a second vector, wherein each vector comprises essentially identical sequences of interest, and wherein the first vector configuration comprises a RBI and a LB1 which are positioned in the first vector to initiate (RB I) and terminate (LB 1) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and wherein the second vector configuration comprises a RB2 and a LB2 which are positioned in the second vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation relative to the sequence of interest in the first T-strand, and wherein the sequence of interest in the two T-strands resulting from initiation at RB I and RB2 are essentially complementary in at least a portion of the sequence of interest.
  • the first vector configuration comprises a RBI and a LB1 which are positioned in the first vector to initiate (RB I) and terminate (LB 1)
  • the Rhizobiales cell comprises at least one vector comprises a first sequence of interest, a second sequence of interest different from the first sequence of interest, at least two RB DNA sequences, and one or more optional LB DNA sequences, wherein the first RB DNA sequence (RBI) and a first LB DNA sequence (LB 1) are positioned in the vector to initiate (RB 1) and terminate (LB 1) synthesis of the first T-strand such that the first sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the vector configuration further comprises a second RB DNA sequence (RB2) and a second LB DNA sequence (LB2) which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the first sequence of interest is in an anti-sense orientation from the 5' to 3' end of the second T-strand, wherein the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other in at least a portion of the first sequence of interest, and
  • the method comprises contacting the plant cell with a first Rhizobiales cell and a second Rhizobiales cell, wherein each Rhizobiales cell contains at least one of two vectors, wherein each vector comprises an essentially identical sequence of interest, and where the first vector comprises a RBI, and wherein the RBI is positioned in the vector to initiate synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the second vector comprises a RB2 which is positioned in the vector to initiate synthesis of a second T-strand such that the sequence of interest is in the anti-sense orientation relative to the sequence of interest in the first T-strand, and wherein the two T-strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the sequence of interest.
  • the method comprises contacting the plant cell with a first Rhizobiales cell and a second Rhizobiales cell, wherein each Rhizobiales cell contains at least one of two vectors, wherein each vector comprises an essentially identical sequence of interest, and where the first vector comprises a RBI and a LB1 which are positioned in the first vector to initiate (RB 1) and terminate (LB 1) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the second vector comprises a RB2 and a LB2 which are positioned in the second vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti- sense orientation from the 5' to 3' end of the second T-strand, and wherein the sequence of interest in the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other.
  • the Rhizobiales cell is selected from an Agrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp., a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp.
  • the Agrobacterium spp. cell is selected from the group consisting of an Agrobacterium
  • the turn efaci ens cell and an Agrobacterium rhizogenes cell in some embodiments, the
  • Rhizobiales cell further contains a vector comprising at least one expression cassette, wherein the expression cassettes comprise a sequence encoding a protein involved in DNA repair, and wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • the method results in at least a fragment of the sequence of interest being integrated into the plant genome by homologous recombination.
  • the method results in at least a fragment of the sequence of interest being integrated into the plant genome by non-homologous end joining.
  • the method further comprises detecting the integration of at least a fragment of the sequence of interest of the vector in the at least one plant cell.
  • the method further comprises selecting the plant cell based on the presence of the at least a fragment of the sequence of interest integrated into the plant genome.
  • the method further comprises regenerating a transgenic plant from the selected plant cell.
  • Several embodiments relate to a method of transforming a plant genome, comprising contacting at least one plant cell on a co-culture medium for at least 24-48 hours, for at least 24-30 hours, for at least 30-36 hours, for at least 36-42 hours, for at least 42-48 hours, for at least 48-54 hours, for at least 54-60 hours, for at least 60-66 hours, for at least 66-72 hours, for at least 72-78 hours, for at least 78-84 hours, for at least 84-90 hours, for at least 90-96 hours, for at least 96-102 hours, for at least 102-108 hours, for at least 108-114 hours, for at least 114-120 hours, for at least 120-126 hours, or for at least 126-132 hours, with at least one Rhizobiales cell capable of transforming the plant cell, wherein the Rhizobiales cell comprises at least one vector comprising a right border (RB) DNA sequence and a left border (LB) DNA sequence and where the vector further comprises between the RB
  • the first sequence of interest further comprises a left homology arm DNA sequence and a right homology arm DNA sequence in and the second sequence of interest further comprises a left homology arm DNA sequence and a right homology arm DNA sequence.
  • the Rhizobiales cell is selected from an Agrobacterium spp., a Bradyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp., a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp.
  • the Agrobacterium spp. cell is selected from the group consisting of an Agrobacterium tumefaciens cell and an Agrobacterium rhizogenes cell. In some
  • the Rhizobiales cell further contains a vector comprising at least one expression cassette, wherein the expression cassettes comprise a sequence encoding a protein involved in DNA repair, and wherein the protein is selected from the group comprising a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • the method results in at least a fragment of the sequence of interest being integrated into the plant genome by homologous recombination. In some embodiments, the method results in at least a fragment of the sequence of interest being integrated into the plant genome by non-homologous end joining.
  • the method further comprises detecting the integration of at least a fragment of the sequence of interest of the vector in the at least one plant cell. In some embodiments, the method further comprises selecting the plant cell based on the presence of the at least a fragment of the sequence of interest integrated into the plant genome. In some embodiments, the method further comprises regenerating a transgenic plant from the selected plant cell.
  • Figures 1A and IB illustrate two control vector configurations in which a sequence of interest is flanked by a right border (RB) DNA sequence on the 5' end, and a left border (LB) DNA sequence on the 3' end (Panel 1A); or alternatively, one RB DNA sequence on the 5' end (Panel IB).
  • RB right border
  • LB left border
  • Figure 1C illustrates a vector configuration comprising two RB DNA sequences, where the first RB DNA sequence (RBI) is positioned in the vector to initiate synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the second RB DNA sequence (RB2) is positioned in the vector to initiate synthesis of a second T-strand such that the sequence of interest is in the anti-sense orientation from the 5' to 3' end of the second T-strand.
  • the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other in at least a portion of the sequence of interest.
  • Figure ID illustrates a vector configuration comprising optional LB DNA sequences.
  • the first RB DNA sequence (RBI) is paired with a first LB DNA sequence (LB 1) which are positioned in the vector to initiate (RB 1) and terminate (LBl) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the second RB DNA sequence (RB2) is paired with a second left border DNA sequence (LB2) which are positioned in the vector to initiate synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation from the 5' to 3' end of the second T-strand.
  • the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other in at least a portion of the sequence of interest.
  • Figure 2 A illustrates a vector configuration comprising a first sequence of interest and a second sequence of interest, where the first sequence of interest is essentially identical to the second sequence of interest; and the vector configuration further comprises a first RB DNA sequence (RBI) with an optional first LB DNA sequence (LBl) which are positioned in the vector to initiate (RB I) and optionally terminate (LBl) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T- strand; and the vector configuration further comprises a second RB DNA sequence (RB2) and an optional second LB DNA sequence (LB2) which are positioned in the vector to initiate (RB2) and optionally terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation from the 5' to 3' end of the second T- strand.
  • the sequence of interest in the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other.
  • Figure 2B illustrates a vector configuration employed in co-transformation in which two essentially identical sequences of interest are located on separate vectors, hosted by one or more bacterium cells, and where the first vector configuration comprises a first RB DNA sequence (RBI) and an optional first LB DNA sequence (LBl) which are positioned in the first vector to initiate (RBI) and optionally terminate (LB l) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T- strand; and the second vector configuration comprises a second RB DNA sequence (RB2) and an optional second LB DNA sequence (LB2) which are positioned in the second vector to initiate (RB2) and optionally terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation from the 5' to 3' end of the second T- strand.
  • the sequence of interest in the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other.
  • Figure 2C illustrates a vector comprising a first sequence of interest, a second sequence of interest different from the first sequence of interest, two or more RB DNA sequences, and one or more optional LB DNA sequences.
  • the illustrated construct is one non-limiting example where the vector configuration comprises a first RB DNA sequence (RBI) and an optional first LB DNA sequence (LBl) which are positioned in the vector to initiate (RBI) and optionally terminate (LBl) synthesis of a first T-strand such that the first sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the vector configuration further comprises a second RB DNA sequence (RB2) and an optional second LB DNA sequence (LB2) which are positioned in the vector to initiate (RB2) and optionally terminate (LB2) synthesis of a second T-strand such that the first sequence of interest is in an anti-sense orientation from the 5' to 3' end of the second T-strand.
  • RBI first RB DNA sequence
  • LBl optional first LB DNA sequence
  • the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other in at least a portion of the first sequence of interest.
  • the vector configuration further comprises a third RB DNA sequence (RB3) and an optional third LB DNA sequence (LB3) which are positioned in the vector to initiate (RB3) and optionally terminate (LB3) synthesis of a third T-strand such that the second sequence of interest is in the sense orientation from the 5' to 3' end of the third T-strand; and the vector configuration further comprises a fourth RB DNA sequence (RB4) and an optional fourth LB DNA sequence (LB4) which are positioned in the vector to initiate (RB4) and optionally terminate (LB4) synthesis of a fourth T-strand such that the second sequence of interest is in an anti-sense orientation from the 5' to 3' end of the fourth T-strand.
  • the two T-strands resulting from initiation at RB3 and RB4 are essentially complementary to each other in at least a portion of the second sequence of interest.
  • Figure 3A illustrates a vector configuration where the vector comprises a RB DNA sequence and a LB DNA sequence and where the vector further comprises between the RB and LB DNA sequences: (i) a first sequence of interest in a sense orientation relative to the RB DNA sequence, (ii) a spacer, and (iii) a second sequence of interest in an anti-sense orientation relative to the RB DNA sequence.
  • the two sequences of interest are essentially complementary, and after synthesis of the T-strand a hairpin structure may form.
  • Figures 3B and 3C illustrate possible variations of at least two cassettes (S-o-1 1 and S-o-1 2) in relation to positions of two homology arms within a sequence of interest.
  • Figure 3B illustrates a first cassette positioned between two homology arms (HA) and a second cassette positioned outside of the one of the homology arms (HA).
  • Figure 3C illustrates both a first cassette and a second cassette positioned between two homology arms (HA).
  • Figure 4A illustrates a schematic of the control vector configuration for vector A used in Example 9.
  • Figure 4B illustrates a schematic of the vector configuration for vector C used in Example 9.
  • Figure 4C illustrates a schematic of the vector configuration for vector D used in Example 9.
  • Figure 5 illustrates the targeted DNA sequence, comprising a left homology arm (HA-L), a selectable marker gene (CP4-EPSPS), and a right homology arm (HA-R), that is used in Example 9.
  • Figure 5 further illustrates the position of PCR primers and Southern blot probes used in Example 9.
  • Figure 6A illustrates a schematic of the configuration of control vector A used in Example 12.
  • Vector A comprises a first sequence of interest comprising an expression cassette encoding CP4-EPSPS positioned between a first RB DNA sequence (RBI) and a first LB DNA sequence (LB1).
  • the vector further comprises a second sequence of interest comprising two expression cassettes each encoding half of a TALEN pair positioned between a second RB DNA sequence (RB2) and a second LB DNA sequence (LB2).
  • Figure 6B illustrates a schematic of the configuration of vector B used in Example 12.
  • the vector comprises a first sequence of interest comprising an expression cassette encoding CP4-EPSPS positioned between two RB DNA sequences and two LB DNA sequences.
  • the vector further comprises a second sequence of interest comprising two expression cassettes each encoding half of a TALEN pair.
  • Figure 7A illustrates a schematic of the configuration of vector A used in Example 13.
  • the vector comprises a first sequence of interest comprising an expression cassette encoding CP4-EPSPS with a TALEN target site (TS) positioned 5' to the CP4-EPSPS cassette. Additionally, the first sequence of interest is flanked by a first RB DNA sequence (RBI) and a second RB DNA sequence (RB2).
  • the vector further comprised a second sequence of interest comprising two expression cassettes each encoding half of a TALEN pair positioned adjacent to a third RB DNA sequence (RB3).
  • Figure 7B illustrates a schematic of the configuration of vector B used in Example 13.
  • the vector comprises a first sequence of interest comprising an expression cassette encoding CP4-EPSPS flanked by TALEN target sites. Additionally, the first sequence of interest is flanked by a first RB DNA sequence (RBI) and a second RB DNA sequence (RB2).
  • the vector further comprised a second sequence of interest comprising two expression cassettes each encoding half of a TALEN pair positioned adjacent to a third RB DNA sequence (RB3).
  • Figure 7C illustrates a schematic of the configuration of vector C used in Example 13.
  • the vector comprises a first sequence of interest comprising an expression cassette encoding CP4-EPSPS positioned between a left homology arm and a right homology arm. Additionally, the first sequence of interest is positioned between a first RB DNA sequence (RBI) and a second RB DNA sequence (RB2).
  • the vector further comprises a second sequence of interest comprising two expression cassettes each encoding half of a TALEN pair positioned adjacent to a third RB DNA sequence (RB3).
  • a cell or tissue culture derived from a plant can comprise any plant components or plant organs (e.g., leaves, stems, roots, etc.), plant tissues, seeds, plant cells, and/or progeny of the same.
  • a progeny plant can be from any filial generation, e.g., Fi, F 2 , F 3 , F 4 , F 5 , F 6 , F 7; etc.
  • a plant cell is a biological cell of a plant, taken from a plant or derived through culture from a cell taken from a plant.
  • transgenic means a plant cell, a plant, a plant part, or a seed whose genome has been altered by the stable integration of exogenous DNA.
  • a transgenic line includes a plant regenerated from an originally-transformed plant cell and progeny transgenic plants from later generations or crosses of a transformed plant.
  • stably transformed is defined as a transfer of DNA into genomic DNA of a targeted cell that allows the targeted cell to pass the transferred DNA to the next generation.
  • the transferred DNA is integrated into the genomic DNA of a reproductive cell.
  • the transferred DNA is integrated into the genomic DNA of a somatic cell.
  • transiently transformed is defined as a transfer of DNA into a cell that is not integrated into the transformed cell's genomic DNA.
  • plant genome refers to a nuclear genome, a mitochondrial genome, or a plastid (e.g., chloroplast) genome of a plant cell.
  • the instant disclosure provides a Rhizobiales cell comprising at least one vector that is capable of forming at least two T-strands that are essentially
  • the instant disclosure provides a Rhizobiales cell comprising at least one vector that is capable of forming at least two T-strands that are essentially complementary for at least a portion of the vector backbone.
  • the instant disclosure provides a Rhizobiales cell comprising at least one vector that is capable of forming two T-strands that are essentially complementary for at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1, 100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2, 100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3, 100, 3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,900, 4,000, 4, 100, 4,200, 4,300, 4,400, 4,500, 4,600, 4,700, 4,800, 4,900, 5,000, 5, 100, 5,200, 5,300, 5,400, 5,500, 5,600, 5,700, 5,800, 5,900, 6,000, 6, 100, 6,200, 6,300, 6,400, 6,500, 6,600, 6,700, 6,800, 6,900, 6,000
  • 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more vectors provided herein are located within a single Rhizobiales cell.
  • the instant disclosure provides a Rhizobiales cell comprising at least a first vector and a second vector, where the first vector is configured to produce a first T-strand comprising a sequence of interest and the second vector is configured to produce a second T-strand comprising a sequence of interest oriented such that the first and second T- strands are essentially complementary in at least a portion of a sequence of interest.
  • the instant disclosure provides a Rhizobiales cell comprising at least a first vector and a second vector that are capable of producing a first T-strand and a second T- strand that are essentially complementary for at least a portion of the vector backbone.
  • the instant disclosure provides a Rhizobiales cell comprising at least a first vector and a second vector that are capable of producing a first T-strand and a second T- strand that are essentially complementary for at least 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2, 100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,900, 4,000, 4,100, 4,200, 4,300, 4,400, 4,500, 4,600, 4,700, 4,800, 4,900, 5,000, 5, 100, 5,200, 5,300, 5,400, 5,500, 5,600, 5,700, 5,800, 5,900, 6,000, 6,100, 6,200, 6,300, 6,400
  • Rhizobiales refers to members of the bacterial Order Rhizobiales that are capable of transforming a plant cell.
  • a Rhizobiales provided herein can refer to an Agrobacterium spp., a Bmdyrhizobium spp., a Mesorhizobium spp., an Ochrobactrum spp., a Phyllobacterium spp., a Rhizobium spp., and a Sinorhizobium spp.
  • a Rhizobiales provided herein can refer to Agrobacterium tumefaciens or Agrobacterium rhizogenes.
  • 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more vectors provided herein are located within a single Rhizobiales cell. In other aspects, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more different vectors provided herein are located within 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more different Rhizobiales cells.
  • recombination refers to the exchange of nucleotides between two nucleic acid molecules.
  • the term “homologous recombination” refers to the exchange of nucleotides at a conserved region shared by two nucleic acid molecules. HR includes symmetric homologous recombination and asymmetric homologous recombination. Asymmetric homologous recombination can also mean unequal recombination.
  • non-homologous end joining HEJ refers to the ligation of two ends of double- stranded DNA without the need of a homologous sequence to direct the ligation.
  • microhomology refers to the presence of the same short sequence (1 to 10 bp) of bases in different nucleic acid molecules.
  • at least one of the nucleic acid molecules is genomic DNA and at least one of the nucleic acid molecules comprises two T-strands that are essentially complementary in at least a portion of a sequence of interest.
  • Methods for detecting HR and NHEJ include, but are not limited to, 1) phenotypic screening, 2) molecular marker technologies such as single nucleotide polymorphism (SNP) analysis by TaqMan® or Illumina/Infinium technology, 3) Southern blot, and 4) sequencing (e.g., Sanger, Illumina®, 454, Pac-Bio, Ion TorrentTM).
  • SNP single nucleotide polymorphism
  • iPCR inverse PCR
  • restriction nuclease sites flanking a targeted gene are identified on each of the two parental chromosomes. These restriction nuclease sites can be the same or different.
  • a PCR primer specific for the first parental chromosome and another PCR primer specific for the second parental chromosome are designed.
  • An induced double-strand break promotes recombination between the two parental chromosomes brings both restriction endonuclease sites and primer binding sites onto the same recombinant chromosome.
  • a PCR product is observed only in instances where recombination occurs.
  • the occurrence of homologous recombination can be detected by PCR, with primers specifically designed for the T-strand insert paired with primers specifically designed for flanking sequences of the target sequence (outside of the homologous regions). For example, when an upstream flanking primer is paired with a downstream insert-specific primer, a PCR product is observed only when recombination occurs.
  • plasmid refers to a circular, double-stranded DNA molecule that is physically separate from chromosomal DNA.
  • a plasmid or vector used herein is capable of replication in vivo.
  • the vector is capable of transforming a plant cell.
  • a plasmid provided herein is a bacterial plasmid.
  • a plasmid provided herein is an Agrobacterium Ti plasmid or derived from an Agrobacterium Ti plasmid.
  • a plasmid or vector provided herein is a recombinant vector.
  • the term "recombinant vector” refers to a vector formed by laboratory methods of genetic recombination, such as molecular cloning.
  • a vector or plasmid provided herein is a synthetic plasmid.
  • a "synthetic plasmid" is an artificially created plasmid that is capable of the same functions ⁇ e.g., replication) as a natural plasmid (e.g., Ti plasmid).
  • one skilled in the art can create a synthetic plasmid de novo via synthesizing a plasmid by individual nucleotides, or by splicing together nucleic acid molecules from different pre-existing plasmids.
  • sequence identity when used in relation to nucleic acids, describe the degree of similarity between two or more nucleotide sequences.
  • the percentage of "sequence identity" between two sequences is determined by comparing two optimally aligned sequences over a comparison window, such that the portion of the sequence in the comparison window may comprise additions or deletions (gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
  • a sequence that is identical at every position in comparison to a reference sequence is said to be identical to the reference sequence and vice- versa.
  • An alignment of two or more sequences may be performed using any suitable computer program. For example, a widely used and accepted computer program for performing sequence alignments is CLUSTALW vl .6 (Thompson, et al. (1994) Nucl. Acids Res., 22: 4673-4680).
  • nucleic acid molecule refers to pairing of nucleotide bases such that A is complementary to T (or U), and G is complementary to C. Two complementary nucleic acid molecules are capable of hybridizing with each other. As an example, the two strands of double stranded DNA are complementary to each other.
  • the term "essentially homologous" or "essentially identical” means that two nucleotide sequences have at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%), at least 99%, or 100% sequence identity with each other.
  • the term "essentially complementary" means that two nucleotide sequences have at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), or 100%) sequence complementarity with each other.
  • a "portion" of a nucleic acid molecule refers to at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of its total length, or at least 5, 10, 15, 20, 25, 30, 25, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1000 or more contiguous nucleotides.
  • polynucleotide refers to a nucleic acid molecule containing multiple nucleotides and generally comprises at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 250, at least 500, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 5000, at least 10,000 or more nucleotide bases.
  • a polynucleotide provided herein can be a plasmid.
  • a specific polynucleotide of 18 - 25 nucleotides in length may be referred to as an
  • oligonucleotide Nucleic acid molecules provided herein include deoxyribonucleic acids (DNA) and ribonucleic acids (RNA) and functional analogues thereof, such as
  • Nucleic acid molecules provided herein can be single stranded or double stranded. Nucleic acid molecules comprise the nucleotide bases adenine (A), guanine (G), thymine (T), cytosine (C). Uracil (U) replaces thymine in RNA molecules.
  • the symbol “N” can be used to represent any nucleotide base (e.g., A, G, C, T, or U).
  • the symbol “K” can be used to represent a G or a T/U nucleotide base.
  • nucleic acid sequences are located on the same nucleic acid molecule.
  • a physical linkage can be adjacent or proximal.
  • a nucleic acid sequence provided herein is adjacent to another nucleic acid sequence.
  • a first nucleic acid molecule provided herein is physically linked to a second nucleic acid molecule provided herein.
  • “flanked” refers to a nucleic acid sequence that is linked on one or both sides to another nucleic acid sequence, including linked to a sequence of interest, or to a LB DNA sequence, or a RB DNA sequence.
  • a flanking sequence precedes a sequence of interest.
  • flanking sequence follows a sequence of interest.
  • a sequence of interest is flanked by another sequence of interest.
  • a flanking sequence is on the 5' or upstream end of a sequence of interest.
  • a flanking sequence is on the 3 ' or downstream end of a sequence of interest.
  • the flanking sequence is contiguous with the sequence of interest.
  • operably linked means that the operably linked nucleic acid sequences exhibit their desired function.
  • a provided DNA promoter sequence can initiate transcription of an operably linked DNA sequence into RNA.
  • a nucleic acid sequence provided herein can be upstream or downstream of a physically or operably linked nucleic acid sequence.
  • a first nucleic acid molecule provided herein is both physically linked and operably linked to a second nucleic acid molecule provided herein.
  • a first nucleic acid molecule provided herein is neither physically linked nor operably linked to a second nucleic acid molecule provided herein.
  • upstream means the nucleic acid sequence is positioned before the 5' end of a linked nucleic acid sequence.
  • downstream means the nucleic acid sequence is positioned after the 3' end of a linked nucleic acid sequence.
  • a "spacer” or a “linker” refers to any polynucleotide sequence capable of forming a loop structure that is at least 4 nucleotides in length. Without being limiting, a spacer provided herein can comprise a non-coding sequence or a coding sequence.
  • T-strand refers to a single-strand copy of DNA made when transcription is initiated from a RB DNA sequence of a vector.
  • synthesis of the transfer DNA is initiated at a 25-base-pair consensus DNA sequence referred to as the "right border” (RB), and T-strand synthesis termination occurs at a 25 bp consensus DNA sequence referred to as the "left border” (LB).
  • RB 25-base-pair consensus DNA sequence
  • LB left border
  • the 25 bp RB consensus DNA sequence (SEQ ID NO: 21) and the 25 bp LB consensus DNA sequence are from the nopaline Agrobacterium tumefaciens strain C58.
  • the 25 bp RB consensus DNA sequence (SEQ ID NO: 22) and the 25 bp LB consensus DNA sequence (SEQ ID NO: 24) are from the octopine Agrobacterium tumefaciens strain A6.
  • a RB consensus DNA sequence is selected from SEQ ID NOs: 21 and 23.
  • a LB consensus DNA sequence is selected from SEQ ID NOs: 22 and 24.
  • a RB DNA sequence or LB DNA sequence comprises at least a 25 bp Ti plasmid RB or LB (respectively) consensus DNA sequence and may additionally comprise a nucleic acid sequence comprising at least 25 nucleotides, at least 50 nucleotides, at least 100 nucleotides, at least 150 nucleotides, at least 200 nucleotides, at least 250 nucleotides, at least 300 nucleotides, at least 350 nucleotides, at least 400 nucleotides, at least 450 nucleotides, at least 500 nucleotides, or at least 600 nucleotides.
  • a RB DNA sequence or a LB DNA sequence provided herein can be of any length such that the DNA segment is capable of transformation of plant tissue performed by Agrobacterium or other Rhizobiales-mediated methods ⁇ See U. S. Patent Nos. 5,731, 179 and 6,265,638; and U.S. Patent Application Publications US2003/110532; US2005/0183170; and US2007/0271627).
  • RB DNA sequences are presented as SEQ ID NOs: 1 - 13 and are variants of A. tumefaciens Ti plasmid sequences ranging from 162 nt to 505 nt in length and comprising either the nopaline RB consensus DNA sequence (SEQ ID NO: 21) or the octopine RB consensus DNA sequence (SEQ ID NO: 22). Similarly, as used in this application, LB DNA sequences are presented as SEQ ID NOs: 14 - 20 and are variants of A.
  • tumefaciens Ti plasmid sequences ranging from 220 nt to 443 nt in length and comprising either the nopaline LB consensus DNA sequence (SEQ ID NO: 23) or the octopine LB consensus DNA sequence (SEQ ID NO: 24).
  • Table 1 contains the SEQ ID NOs for each of the RB and LB DNA sequences presented herein with the position indicated within each SEQ ID NO of the nopaline or octopine 25 bp consensus sequences. Other RB and LB sequences are contemplated. Table 1.
  • a vector provided herein can comprise at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 RB DNA sequences.
  • the RB DNA sequence comprises an octopine Agrobacterium Ti plasmid 25 bp RB DNA consensus sequence.
  • the RB DNA sequence comprises a nopaline Agrobacterium Ti plasmid 25 bp RB consensus DNA sequence.
  • the RB DNA sequences may be essentially homologous or the RB DNA sequences may not be essentially homologous.
  • the one or more RB DNA sequences comprise a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to a sequence selected from SEQ ID NOs: 1-13.
  • the first RB (RBI) DNA sequence or the second RB (RB2) DNA sequence comprises a sequence selected from the group comprising SEQ ID NOs: 1-13.
  • the RBI DNA sequence comprises a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%), at least 97%, at least 98%, at least 99% identical, or 100% identical to a sequence selected from SEQ ID NO: 4 or SEQ ID NO: 12; and the RB2 DNA sequence comprises a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to a sequence
  • the at least one vector disclosed herein does not comprise a LB DNA sequence.
  • the at least one vector disclosed herein comprises at least one, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 LB DNA sequences.
  • the LB DNA sequences may be essentially homologous or the LB DNA sequences may not be essentially homologous.
  • the LB DNA sequence comprises an octopine Agrobacterium Ti plasmid 25 bp LB consensus DNA sequence.
  • the LB DNA sequence comprises a nopaline Agrobacterium Ti plasmid 25 bp LB consensus DNA sequence.
  • the one or more of the LB DNA sequences are selected from the group comprising SEQ ID NOs: 14-20.
  • the two or more LB DNA sequences comprise a sequence at least 80%, at least 81%, at least 81%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to a sequence selected from SEQ ID NOs: 14-20.
  • the LB DNA sequence comprises a sequence at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical, or 100% identical to SEQ ID NO: 19.
  • sequence of interest refers to a polynucleotide sequence in a vector that forms part of the T-strand.
  • a vector provided herein comprises 0, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10, or more sequences of interest.
  • a sequence of interest in a vector disclosed herein does not include RB or a LB DNA sequences, and does not include vector backbone sequence.
  • vector configurations with two sequences of interest positioned between two RB DNA sequences such that the sequence of interest in the two T- strands synthesized from the two RB DNA sequences are essentially complementary.
  • the nucleotide sequence of the two sequences of interest may be identical, or the nucleotide sequence may be at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
  • a sequence of interest provided herein comprises 0, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 expression cassettes.
  • the sequence of interest provided herein comprises one or more expression cassettes physically and/or operably linked in a cassette stack.
  • a sequence of interest comprises an expression cassette adjacent to a left homology arm DNA sequence, a right homology arm DNA sequence, or a left homology arm DNA sequence and a right homology arm DNA sequence.
  • a sequence of interest comprises an expression cassette flanked by homology arm DNA sequences.
  • a sequence of interest comprises one ore more expression cassettes that is not flanked by homology arms.
  • a sequence of interest comprises one or more expression cassettes flanked by one ore more site-specific enzyme target sites. In some aspects, a sequence of interest comprises one or more expression cassettes flanked by one ore more recombinase recognition sites. In another aspect, a sequence of interest provided herein comprises an endogenous polynucleotide sequence. In some embodiments, the endogenous polynucleotide sequence comprises an intergenic sequence, a native gene, or a mutated gene. In another aspect, a sequence of interest provided herein comprises an exogenous
  • At least part of the sequence of interest in the vector disclosed herein is integrated into a plant genome via HR. In another aspect, at least part of the sequence of interest in the vector disclosed herein is integrated into a plant genome via HEJ.
  • a sequence of interest is flanked by at least two homology arm DNA sequences and comprises a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%), at least 98%, at least 99%, or 100% identical to a native gene of a plant cell.
  • the at least two homology are sequences are flanked by one or more site- specific enzyme target sites.
  • the at least two homology are sequences are flanked by one or more recombinase recognition sites.
  • a sequence of interest provided herein comprises 0, at least 1, or at least 2 homology arm DNA sequences.
  • a sequence of interest provided herein comprises at least two homology arm DNA sequences the at least two homology arm DNA sequences can be distinguished by referring to them as a "left homology arm DNA sequence" and a "right homology arm DNA sequence.”
  • a sequence of interest provided herein comprises both a left homology arm DNA sequence and a right homology arm DNA sequence.
  • a right homology arm DNA sequence and a left homology arm DNA sequence provided herein are homologous to a targeted genomic DNA sequence in the plant or plant cell.
  • a right homology arm DNA sequence and a left homology arm DNA sequence are not essentially homologous to each other.
  • a right homology arm DNA sequence and a left homology arm DNA sequence are essentially homologous to each other.
  • a sequence of interest comprises one or more expression cassettes positioned between a right homology arm DNA sequence and a left homology arm DNA sequence.
  • a sequence of interest comprises a sequence for templated genome editing positioned between a right homology arm DNA sequence and a left homology arm DNA sequence.
  • at least part of a sequence of interest provided herein is outside of the region comprising a left homology arm DNA sequence, a right homology arm DNA sequence, and one or more cassettes.
  • at least part of a sequence of interest provided herein is within the region comprising a left homology arm DNA sequence, a right homology arm DNA sequence, and a sequence for templated genome editing.
  • a sequence of interest provided herein comprises a first sequence positioned between a left homology arm DNA sequence and a right homology arm DNA sequence, and a second sequences that is not positioned between a left homology arm DNA sequence and a right homology arm DNA sequence.
  • a vector provided herein comprises a first sequence of interest further comprising a first left homology arm DNA sequence and a first right homology arm DNA sequence and a second sequence of interest further comprising a second left homology arm DNA sequence and a second right homology arm DNA sequence.
  • a sequence of interest provided herein is integrated into a plant genome in its entirety. In an aspect, at least a part of a sequence of interest provided herein is integrated into a plant genome. In another aspect, only the sequence of interest between a right homology arm DNA sequence and a left homology arm DNA sequence is integrated into a plant genome. In another aspect, a sequence of interest provided herein is integrated into a plant genome via HR. In an aspect, HR can occur between one or two homology arm DNA sequences provided herein and a plant genome. In another aspect, a sequence of interest provided herein is integrated into a plant genome via NHEJ. In another aspect, a sequence of interest provided herein is integrated into a plant genome via microhomology-mediated end joining. In yet another aspect, a sequence of interest provided herein is randomly integrated into a plant genome via Agrobacterium-mediated T-strand integration.
  • integration of at least part of a sequence of interest provided herein results in one or more point mutations, one or more insertions, one or more deletions, an inversion, increased transcription of an endogenous locus, decreased transcription of an endogenous locus or any combination thereof.
  • integration of at least part of a sequence of interest provided herein results in altered protein activity, altered production of RNAi polynucleotides, altered sequence of RNAi target sites, altered RNAi pathway activity, increased transcription of the sequence of interest, silencing of the integrated sequence of interest, or any combination thereof.
  • Silencing technologies include, without limitations, antisense-, co-suppression-mediated mechanisms, and RNAi technologies, such as miRNA (e.g., U. S. Patent Application Publication 2006/0200878).
  • integration of at least part of a sequence of interest causes targeted transcription, decreased transcription, enhanced transcription, or templated editing of a transgenic nucleic acid sequence present in the genome.
  • homology arm or “homology arm DNA sequence” refers to a polynucleotide sequence that has at least 80%, at least 85%>, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%) or 100%> sequence identity to a target sequence in a plant or plant cell.
  • a homology arm can comprise at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 500, at least 550, at ;east 600, at least 650, at least 700, at least, 750, at least 800, at least 850, at least 900, at least 950, at least 1000, or at least 2500 nucleotides.
  • the target sequence comprises a protein-coding sequence.
  • the target sequence is a genie sequence.
  • a "genie" sequence is a nucleic acid sequence that encodes a protein or a non-protein-coding RNA.
  • a genie sequence can include one or more introns.
  • the target sequence is a non-genic sequence.
  • a "non-genic" sequence is a nucleic acid sequence that is not a genie sequence.
  • the target sequence comprises a non-coding sequence.
  • the target sequence comprises both a protein-coding sequence and a non- coding sequence.
  • the target sequence does not comprise a gene or a portion of a gene.
  • the target sequence is linked to a gene of interest.
  • the target sequence is linked to a transgene integrated in the genome of a plant or plant cell.
  • target sequence refers to a genomic locus selected for targeted integration of a sequence of interest.
  • the sequence of interest is integrated into the target sequence by HR or NHEJ.
  • target sequence can refer to the full-length nucleotide sequence of the genomic locus targeted for cleavage and recombination, or the nucleotide sequence of a portion of the genomic locus targeted for cleavage and recombination.
  • the target sequence can be an endogenous genomic locus or a transgene.
  • a target sequence is a genie sequence.
  • a target sequence is a non-genic sequence.
  • a "site-specific recombination site” or “site-specific target site” are used interchangeably to refer to a nucleic acid sequence where exogenous DNA is inserted by HR or by non-homologous recombination.
  • site-specific enzyme target site refers to the site that is cleaved by a nuclease introducing a double stranded break into the nucleic acid backbone. The site of the double-strand break may be a target site for introduction of a sequence of interest.
  • endogenous refers to a nucleic acid sequence that exists naturally in the genome of a cell.
  • a method provided herein is used to modify an endogenous locus so that the modified locus shares at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, or at least 99.9% sequence identity as compared to an unmodified endogenous locus.
  • an endogenous nucleic acid sequence undergoes "template editing.” Template editing can occur via HR between a target sequence and a donor template after a double-stranded break occurs in or near the target sequence.
  • a "donor template” is a nucleic acid sequence that shares at least 60% at least 60%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%), or at least 99.9% sequence identity to at least a portion of a target sequence.
  • a donor template provided herein comprises a sequence of interest.
  • Template editing can introduce one or more point mutations, deletions, or insertions into a target sequence.
  • the entire donor template, a portion of the donor template, a copy of the donor template, or a portion of a copy of the donor template integrates into the target sequence.
  • template editing would be analogous to an orthologous nucleic acid sequence, a paralogous nucleic acid sequence, an isogenic nucleic acid sequence, or a cisgenic nucleic acid sequence of the endogenous genome.
  • exogenous refers to a nucleic acid sequence that is not normally present in a cell, but can be introduced into a cell by one or more genetic, biochemical or other methods.
  • an exogenous nucleic acid sequence can be
  • cassette or "expression cassette” refer to a nucleic acid sequence which may or may not be operably linked to one or more expression elements such as an enhancer, a promoter, a leader, an intron, a 5' untranslated region (UTR), a 3' UTR, or a transcription termination sequence.
  • expression elements such as an enhancer, a promoter, a leader, an intron, a 5' untranslated region (UTR), a 3' UTR, or a transcription termination sequence.
  • a cassette comprises a nucleic acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), or 100%) identical to an endogenous nucleic acid sequence.
  • the cassette comprises an exogenous nucleic acid sequence.
  • a cassette in a vector disclosed herein comprises at least one sequence selected from: a gene, a portion of a gene, an intergenic sequence, an enhancer, a promoter, an intron, an exon, a transcription termination sequence, a sequence encoding a chloroplast targeting peptide, a sequence encoding a mitochondrial targeting peptide, an insulator sequence, a sequence encoding an anti-sense RNA, a sequence encoding non-protein-coding RNA (npcRNA), a sequence encoding a recombinase, a sequence encoding a recombinase recognition site (for example, a lox site or a flp site), a sequence encoding an Argonaute (non-limiting examples of Argonaute proteins include Thermus thermophilics Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium gregoryi
  • the cassette is positioned between a first right border (RBI) and a second right border (RB2) such that the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other in at least a portion of the cassette.
  • the cassette is positioned adjacent to at least one LB DNA sequence.
  • the cassette is not positioned between RB DNA sequences.
  • the cassette is not positioned between a RB DNA sequence and a LB DNA sequence.
  • the term "landing pad" refers to a nucleic acid locus that is designed to be the locus for site-specific recombination.
  • landing pads may comprise one or more nucleic acid sequences that are not homologous to native sequences of the host organism. In some embodiments, landing pads may comprise one or more recognition sites for any of an endonuclease, a recombinase or a transposase. In some embodiments a landing pad may comprise one or more recognition sites for any of an endonuclease, a recombinase or a transposase flanking one or more nucleotide sequences substantially lacking homology with the genome of the host organism.
  • the one or more recognition sites are binding sites for DNA-binding domains (e.g., zinc finger proteins (ZFPs), meganucleases, or leucine zippers).
  • landing pads may comprise nucleotide sequences that have substantially no homology to regions of any sequenced target plant genome.
  • the landing pad may comprises any combination of one or more Zinc Finger Nuclease recognition sites, one or more
  • an editing template refers to a nucleic acid sequence that can be used for recombination with a target sequence.
  • an editing template comprises a sequence that is at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% identical to at least a portion of a target sequence.
  • the editing template comprises one or more nucleic acid changes compared to the target sequence.
  • cassette stack refers to two or more expression cassettes which are physically linked in a vector. In some embodiments, two or more cassettes in a cassette stack are operably linked. In some embodiments, two or more cassettes in a cassette stack are not operably linked. In some embodiments, two or more cassettes in a cassette stack are flanked by one or more recombinase recognition sites. In some embodiments, two or more cassettes in a cassette stack are flanked by one or more a site-specific enzyme target sites.
  • two or more cassettes in a cassette stack may be separated by spacer sequences, insulator sequences, multiple cloning site sequences, one or more recombinase recognition sites, a sequence encoding a site-specific enzyme target site, a landing pad, homology arms, a RB DNA sequence or a LB DNA sequence.
  • the cassette stack is positioned between a first right border (RBI) and a second right border (RB2) such that the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other in at least a portion of the cassette stack.
  • the cassette stack is positioned adjacent to at least one LB DNA sequence.
  • the cassette stack is not positioned between RB DNA sequences.
  • the cassette stack is not positioned between a RB DNA sequence and a LB DNA sequence.
  • genomic locus means a locatable region of genomic sequence, corresponding to a unit of inheritance.
  • a genomic locus may comprise one or more regulatory regions, such as promoters, enhancers, 5' UTRs, intron regions, 3'UTRs, transcribed regions, and other functional sequence regions that may exist as native genes or transgenes in a plant or a mammalian genome.
  • gene refers to a sequence that encodes a protein, or a sequence encoding a non-protein-coding RNA.
  • protein-coding refers to a
  • polynucleotide encoding for the amino acids of a polypeptide.
  • "encoding” refers to a polynucleotide that can produce a functional unit (without being limiting, for example, a protein, a microRNA, a transfer RNA, a ribosomal RNA, a small interfering RNA, a guide RNA, a tracer RNA, a single-guide RNA) via transcription and/or translation.
  • a series of three nucleotide bases encodes one amino acid.
  • “expressed,” “expression,” or “expressing” refers to transcription of RNA from a DNA molecule.
  • a sequence of interest provided herein comprises a protein-coding sequence.
  • npcRNA refers to non-protein-coding RNA.
  • Non- limiting examples of non-protein-coding RNA include a microRNA (miRNA), a miRNA precursor, a small interfering RNA (siRNA), a small RNA (22-26 nt in length) and precursor encoding same, a heterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), a hairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA (ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a tracer RNA (tcRNA), a guide RNA (gRNA), and a single-guide RNA (sgRNA).
  • a sequence of interest provided herein comprises a non-protein-coding RNA.
  • an expression cassette comprises at least one gene selected from a gene of agronomic interest, a DNA binding gene, a selectable marker gene, an RNAi construct, a site specific nuclease gene, a recombinant guide RNA of an RNA-guided nuclease
  • RNA-guided nucleases include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl
  • a gene provided herein comprises a promoter. In another aspect, a gene provided herein does not comprise a promoter.
  • genes for disease, insect, or pest tolerance for example, virus tolerance, bacteria tolerance, fungus tolerance, nematode tolerance, arthropod tolerance, gastropod tolerance
  • herbicide tolerance genes for quality improvements such as yield, nutritional enhancements, environmental or stress tolerances, or any desirable changes in plant physiology, growth, development, morphology or plant product(s) including starch production (U.S. Pat. Nos. 6,538, 181; 6,538,179; 6,538,178; 5,750,876; 6,476,295), modified oils production (U.S. Pat. Nos.
  • an expression cassette provided herein comprises a nucleic acid sequence selected from an insecticidal resistance gene, an herbicide tolerance gene, a nitrogen use efficiency gene, a water use efficiency gene, a nutritional quality gene, a DNA binding gene, a selectable marker gene, an RNAi construct, a site specific nuclease gene, a guide RNA, and any combination thereof.
  • a gene can also include polynucleotide sequences that encode for other polynucleotide sequences such as a messenger RNA (mRNA).
  • mRNA messenger RNA
  • An mRNA produced from a nucleic acid molecule of this disclosure can contain a 5'-UTR leader sequence.
  • This sequence can be derived from the promoter selected to express the gene and can be specifically modified so as to increase or decrease translation of the mRNA.
  • a 5'-UTR can also be obtained from viral RNAs, from suitable eukaryotic genes, or from a synthetic gene sequence. Such "enhancer" sequences can be desirable to increase or alter the translational efficiency of the resultant mRNA. This disclosure is not limited to constructs where the non- translated region is derived from the 5'-UTR that accompanies the promoter sequence.
  • the 5'-UTR sequence can be derived from unrelated promoters or genes (see, for example U.S. Pat. No. 5,362,865).
  • non-translation leader sequences include maize and petunia heat shock protein leaders (U.S. Pat. No. 5,362,865), plant virus coat protein leaders, plant rubisco leaders, GmHsp (U.S. Pat. No. 5,659,122), PhDnaK (U.S. Pat. No. 5,362,865), AtAntl, TEV (Carrington and Freed, Journal of Virology, (1990) 64: 1590- 1597), and AGRtu.nos (GenBank Accession V00087; Bevan et al. , Nucleic Acids Research (1983) 11 :369-385).
  • Other genetic components that serve to enhance expression or affect transcription or translational of a gene are also envisioned as genetic components.
  • a gene can further comprise a 3'-UTR.
  • the provided 3'-UTRs can contain a transcriptional terminator, or an element having equivalent function, and a polyadenylation signal that functions in plants to cause the addition of polyadenylated nucleotides to the 3' end of an RNA molecule.
  • suitable 3' regions are (1) the 3' transcribed, non- translated regions containing the polyadenylation signal of Agrobacterium Ti plasmid genes, such as the nopaline synthase (NOS; Fraley et al., Proceedings of the National Academy of Sciences, USA (1983) 80: 4803-4807) gene, and (2) plant genes such as the soybean storage protein genes and the small subunit of the ribulose-l,5-bisphosphate carboxylase
  • ssRUBISCO ssRUBISCO gene.
  • An example of a 3' region is that from the ssRUBISCO E9 gene from pea (European Patent Application 0385 962).
  • an expression cassette or sequence of interest provided herein may comprise a sequence encoding a cell factor that functions to increase DNA repair, where the protein is selected from the group consisting of a vir gene from the Ti plasmid, Rad51, Rad52, Rad2, a dominant-negative Ku70, or any combination thereof.
  • an expression cassette provided herein comprises a nucleic acid sequence that is not essentially homologous to an endogenous plant nucleic acid sequence.
  • an expression cassette provided herein comprises a nucleic acid sequence that is not essentially homologous to an endogenous plant gene.
  • an expression cassette provided herein comprises a nucleic acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an endogenous plant gene.
  • an expression cassette provided herein comprises a nucleic acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%), or 100%) identical to an endogenous plant nucleic acid sequence.
  • a promoter contains a sequence of nucleotide bases that signals RNA polymerase to associate with the DNA and to initiate transcription into mRNA using one of the DNA strands as a template to make a corresponding complementary strand of RNA.
  • a promotor provided herein is a constitutive promoter.
  • a promoter provided herein is a regulatable promoter.
  • an expression cassette provided herein can comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 promoters.
  • a promoter provided herein is located within a sequence of interest. In another aspect, a promoter provided herein is not located within a sequence of interest.
  • promoters that are active in plant cells have been described in the literature. Such promoters would include but are not limited to the nopaline synthase (NOS) and octopine synthase (OCS) promoters that are carried on Ti plasmids of Agrobacterium tumefaciens, the caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S and 35S promoters and the Figwort mosaic virus (FMV) 35S promoter, and the enhanced CaMV35S promoter (e35S).
  • NOS nopaline synthase
  • OCS octopine synthase
  • CaMV cauliflower mosaic virus
  • FMV Figwort mosaic virus
  • e35S enhanced CaMV35S promoter
  • a promoter is capable of causing sufficient expression to result in the production of an effective amount of the gene product of interest. Examples describing such promoters include without limitation U.S. Pat. No. 6,437,217 (maize RS81 promoter), U.S. Pat. No. 5,641,876 (rice actin promoter), U.S. Pat. No. 6,426,446 (maize RS324 promoter), U.S. Pat. No.
  • 6,429,362 (maize PR-1 promoter), U.S. Pat. No. 6,232,526 (maize A3 promoter), U.S. Pat. No. 6, 177,611 (constitutive maize promoters), U.S. Pat. Nos. 5,322,938, 5,352,605, 5,359,142 and 5,530,196 (35S promoter), U.S. Pat. No. 6,433,252 (maize L3 oleosin promoter), U.S. Pat. No. 6,429,357 (rice actin 2 promoter as well as a rice actin 2 intron), U.S. Pat. No. 5,837,848 (root specific promoter), U.S. Pat. No.
  • a nopaline synthase (NOS) promoter (Ebert et al., 1987), the octopine synthase (OCS) promoter (which is carried on tumor-inducing plasmids of Agrobacterium tumefaciens), the caulimovirus promoters such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al, Plant Molecular Biology (1987) 9: 315-324), the CaMV 35S promoter (Odell et al, Nature (1985) 313 : 810-812), the figwort mosaic virus 35S-promoter (U.S. Pat. Nos.
  • NOS nopaline synthase
  • OCS octopine synthase
  • sucrose synthase promoter (Yang and Russell, Proceedings of the National Academy of Sciences, USA ( 1990) 87: 4144-4148), the R gene complex promoter (Chandler et al, Plant Cell (1989) 1 : 1175-1183), and the chlorophyll a/b binding protein gene promoter, PC1 SV (U.S. Pat. No. 5,850,019), and AGRtu.nos (GenBank Accession V00087; Depicker et al., Journal of Molecular and Applied Genetics (1982) 1 : 561-573; Bevan et al., 1983) promoters.
  • promoter hybrids can be constructed to enhance transcriptional activity (U.S. Pat. No. 5,106,739), or to combine desired transcriptional activity, inducibility and tissue specificity or developmental specificity.
  • Promoters that function in plants include but are not limited to promoters that are inducible, viral, synthetic, constitutive, temporally regulated, spatially regulated, and spatio-temporally regulated.
  • Other promoters that are tissue-enhanced, tissue-specific, or developmentally regulated are also known in the art and envisioned to have utility in the practice of this disclosure.
  • Promoters used in the provided nucleic acid molecules and vectors of this disclosure can be modified, if desired, to affect their control characteristics. Promoters can be derived by means of ligation with operator regions, random or controlled mutagenesis, etc. Furthermore, the promoters can be altered to contain multiple "enhancer sequences" to assist in elevating gene expression.
  • a sequence of interest provided herein can comprise one or more selectable or screenable marker genes.
  • the selectable or screenable marker gene aids in the identification of a transformed plant, or a product of agronomic utility.
  • DNA that serves as a selectable or screenable marker can function in a regenerable plant tissue to produce a compound that would confer upon the plant tissue resistance to an otherwise toxic compound.
  • selectable or screenable marker genes are known in the art and can be used.
  • Genes for use as a selectable or screenable marker can include, but are not limited, to ⁇ -glucuronidase (GUS), green fluorescent protein (GFP), luciferase (LUC), genes conferring tolerance to antibiotics like kanamycin (Dekeyser et al., Plant Physiology (1989) 90: 217-223) or spectinomycin (e.g. spectinomycin aminoglycoside adenyltransferase (aadA); U.S. Pat. No. 5,217,902), genes that encode enzymes that give tolerance to herbicides like glyphosate (e.g.
  • EPSPS 5- enolpymvylshikimate-3 -phosphate synthase
  • sulfonyl herbicides e.g. acetohydroxyacid synthase or acetolactate synthase conferring tolerance to acetolactate synthase inhibitors such as sulfonylurea, imidazolinone, triazolopyrimidine, pyrimidyloxybenzoates and phthalide; (U.S. Pat. Nos. 6,225,105;
  • Escherichia coli allowing growth in the presence of mannose), and dual selection (e.g.
  • spectinomycin at a concentration of about 25-1000 ppm, such as at about 150 ppm, is also contemplated.
  • a selectable or screenable marker provided herein is a positive selection marker.
  • a positive selection marker confers an advantage to a cell comprising such marker.
  • a sequence of interest provided herein comprises one or more positive selection markers.
  • a positive selection marker confers antibiotic resistance or herbicide resistance.
  • a selectable or screenable marker provided herein is a negative selection marker.
  • a sequence of interest provided herein comprises one or more negative selection markers.
  • a selectable or screenable marker provided herein is both a positive selection marker and a negative selection marker.
  • a negative selectable marker provided herein can be a lethal or non-lethal negative selectable marker.
  • non-lethal negative selectable markers examples include U.S. Publication No. 2004-0237142, such as GGPP synthases, GA 2-oxidase gene sequences, isopentenyl transferase (IPT), CKI1 (cytokinin-independent 1), ESR-2, ESR1-A, auxin-producing genes, such as indole-3-acetic acid (IAA), iaaM, iaah, roLABC, genes that result in over-expression of ethylene biosynthetic enzymes, VP1 genes, AB13 genes, LEC1 genes, and Basl genes for example.
  • a non-lethal negative selectable marker gene can be included on any nucleic acid molecule provided herein.
  • a non-lethal negative selectable marker gene provided herein is a gene resulting in the over-expression of a class of enzymes that use substrates of the gibberellic acid (GA) biosynthetic pathway, but that do not result in the production of bioactive GA.
  • a nucleic acid molecule provided herein comprises a non-lethal negative selectable marker gene such as a phytoene synthase gene from Erwinia herbicola (crtB).
  • the selectable or screenable marker imparts a distinct phenotype to cells expressing the marker protein and often provide a means to more efficiently distinguish such transformed cells from cells that do not have the selectable or screenable marker.
  • Such genes may encode either a selectable or screenable marker, depending on whether the marker confers a trait which one can "select" for by chemical means, such as through the use of a selective agent (for example, an herbicide, or an antibiotic), or whether it is simply a trait that one can identify through observation (for example, expression of GFP) or testing or "screening".
  • a selective agent for example, an herbicide, or an antibiotic
  • suitable marker proteins are known to the art and can be employed in the practice of the invention.
  • selectable or “screenable markers” also are genes which encode a "secretable marker” whose secretion can be detected as a means of identifying or selecting for transformed cells. Examples include markers which are secretable antigens that can be identified by antibody interaction, or even secretable enzymes which can be detected by their catalytic activity. Secretable proteins fall into a number of classes, including small, diffusible proteins detectable, by ELISA; or small active enzymes detectable in extracellular solution (for example, a-amylase, ⁇ -lactamase, phosphinothricin
  • proteins that are inserted or trapped in the cell wall for example, proteins that include a leader sequence such as that found in the expression unit of extensin or tobacco PR-S.
  • screenable markers examples include a
  • ⁇ -glucuronidase GUS or uidA gene which encodes an enzyme for which various chromogenic substrates are known
  • an R-locus gene which encodes a product that regulates the production of anthocyanin pigments (red color) in plant tissues (Dellaporta et al, 1988); a ⁇ -lactamase gene (Sutcliffe, 1978), which encodes an enzyme for which various chromogenic substrates are known (e.g., PAD AC, a chromogenic cephalosporin); a y/E gene (Zukowsky et al, 1983) which encodes a catechol dioxygenase that can convert chromogenic catechols; an ⁇ -amylase gene (Ikuta et al, 1990); a tyrosinase gene (Katz et al, 1983) which encodes an enzyme capable of oxidizing tyrosine to DOPA and dopaquinone which in turn condenses to
  • a sequence of interest provided herein comprises polynucleotides encoding at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 site-specific enzymes.
  • a plant cell provided herein already comprises a polynucleotide encoding a site-specific enzyme.
  • a polynucleotide encoding a site-specific enzyme provided herein is stably transformed into a plant cell.
  • a polynucleotide encoding a site-specific enzyme provided herein is transiently transformed into a plant cell.
  • a polynucleotide encoding a site-specific enzyme is under the control of a regulatable promoter, a constitutive promoter, a tissue specific promoter, or any promoter useful for expression of the site- specific enzyme.
  • a vector comprises in cis a cassette encoding a site-specific enzyme and a sequence of interest such that when contacted with the genome of a plant cell, the site-specific enzyme enables site-specific integration of the sequence of interest.
  • a first vector comprises a cassette encoding a site-specific enzyme and a second vector comprises a sequence of interest such that when contacted with the genome of a plant cell, the site-specific enzyme provided in trans enables site-specific integration of the sequence of interest.
  • site-specific enzyme refers to any enzyme that can cleave a nucleotide sequence in a site-specific manner.
  • a site-specific enzyme provided herein is selected from the group consisting of an endonuclease (without being limiting, for example, a meganuclease, a zinc-finger nuclease (ZFN), a transcription activator-like effector nucleases (TALEN), an Argonaute (non-limiting examples of
  • Argonaute proteins include Thermus thermophilics Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium gregoryi Argonaute (NgAgo), an RNA-guided nuclease (non-limiting examples of RNA-guided nucleases include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Cs
  • a tyrosine recombinase attached to a DNA recognition motif provided herein is selected from the group consisting of a Cre recombinase, a Gin
  • a Cre recombinase or a Gin recombinase provided herein is tethered to a zinc-finger DNA binding domain.
  • a serine recombinase attached to a DNA recognition motif provided herein is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase.
  • a DNA transposase attached to a DNA binding domain provided herein is selected from the group consisting of a TALE-piggyBac and TALE-Mutator.
  • Site-specific nucleases such as meganucleases, ZFNs, TALENs, Argonaute proteins
  • Argonaute proteins include Thermus thermophilics Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium gregoryi Argonaute (NgAgo), homologs thereof, or modified versions thereof
  • Cas9 nucleases Non- limiting examples of RNA-guided nucleases include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl,
  • Csf2, Csf3, Csf4, Cpfl, CasX, CasY, homologs thereof, or modified versions thereof induce a double-strand DNA break at the target site of a genomic sequence that is then repaired by the natural processes of HR or NHEJ. Sequence modifications then occur at the cleaved sites, which can include deletions or insertions that result in gene disruption in the case of NHEJ, or integration of nucleic acid sequences by HR.
  • a vector or sequence of interest provided herein can comprise a nucleic acid sequence encoding a zinc finger nuclease.
  • ZFNs are synthetic proteins consisting of an engineered zinc finger DNA-binding domain fused to the cleavage domain of the Fokl restriction endonuclease.
  • ZFNs can be designed to cleave almost any long stretch of double- stranded DNA for modification of the zinc finger DNA-binding domain.
  • ZFNs form dimers from monomers composed of a non-specific DNA cleavage domain of Fokl endonuclease fused to a zinc finger array engineered to bind a target DNA sequence.
  • the DNA-binding domain of a ZFN is typically composed of 3-4 zinc-finger arrays.
  • the amino acids at positions -1, +2, +3, and +6 relative to the start of the zinc finger co-helix, which contribute to site-specific binding to the target DNA, can be changed and customized to fit specific target sequences.
  • the other amino acids form the consensus backbone to generate ZFNs with different sequence specificities. Rules for selecting target sequences for ZFNs are known in the art.
  • the Fokl nuclease domain requires dimerization to cleave DNA and therefore two ZFNs with their C-terminal regions are needed to bind opposite DNA strands of the cleavage site (separated by 5-7 bp).
  • the ZFN monomer can cut the target site if the two-ZF- binding sites are palindromic.
  • ZFN as used herein, is broad and includes a monomelic ZFN that can cleave double stranded DNA without assistance from another ZFN.
  • the term ZFN is also used to refer to one or both members of a pair of ZFNs that are engineered to work together to cleave DNA at the same site.
  • a vector or sequence of interest provided herein can comprise a nucleic acid sequence encoding a transcription activator-like effector nuclease (TALEN).
  • TALENs are artificial restriction enzymes generated by fusing the transcription activator-like effector (TALE) DNA binding domain to a nuclease domain.
  • the nuclease is selected from a group consisting of PvuII, MutH, TevI and Fokl, Alwl, Mlyl, Sbfl, Sdal, Stsl, CleDORF, Clo051, PeptOVl.
  • TALEN as used herein, is broad and includes a monomelic TALEN that can cleave double stranded DNA without assistance from another TALEN.
  • TALEN is also used to refer to one or both members of a pair of TALENs that work together to cleave DNA at the same site.
  • Transcription activator-like effectors can be engineered to bind practically any DNA sequence.
  • TALE proteins are DNA-binding domains derived from various plant bacterial pathogens of the genus Xanthomonas . The X pathogens secrete TALEs into the host plant cell during infection.
  • TALE moves to the nucleus, where it recognizes and binds to a specific DNA sequence in the promoter region of a specific DNA sequence in the promoter region of a specific gene in the host genome.
  • TALE has a central DNA-binding domain composed of 13-28 repeat monomers of 33-34 amino acids.
  • the amino acids of each monomer are highly conserved, except for hypervariable amino acid residues at positions 12 and 13.
  • the two variable amino acids are called repeat-variable diresidues (RVDs).
  • RVDs repeat-variable diresidues
  • the amino acid pairs NI, NG, HD, and NN of RVDs preferentially recognize adenine, thymine, cytosine, and guanine/adenine, respectively, and modulation of RVDs can recognize consecutive DNA bases. This simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs.
  • Fokl domains Besides the wild-type Fokl cleavage domain, variants of the Fokl cleavage domain with mutations have been designed to improve cleavage specificity and cleavage activity.
  • the Fokl domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALEN DNA binding domain and the Fokl cleavage domain and the number of bases between the two individual TALEN binding sites are parameters for achieving high levels of activity.
  • PvuII, MutH, and Tevl cleavage domains are useful alternatives to Fokl and Fokl variants for use with TALEs.
  • PvuII functions as a highly specific cleavage domain when coupled to a TALE (see Yank et al. 2013. PLoS One. 8: e82539). MutH is capable of introducing strand-specific nicks in DNA (see Gabsalilow et al. 2013. Nucleic Acids Research. 41 : e83). Tevl introduces double-stranded breaks in DNA at targeted sites(see Beurdeley et al., 2013. Nature Communications. 4: 1762).
  • TALE binding domain allows for designable proteins.
  • Software programs such as DNA Works can be used to design TALE constructs.
  • Other methods of designing TALE constructs are known to those of skill in the art. See Doyle et al, Nucleic Acids Research (2012) 40: Wl 17-122.; Cermak et al., Nucleic Acids Research (2011). 39:e82; and talent, cac. Cornell . edu/ about.
  • a vector or sequence of interest provided herein can comprise a nucleic acid sequence encoding a meganuclease.
  • Meganucl eases which are commonly identified in microbes, are unique enzymes with high activity and long recognition sequences (> 14 bp) resulting in site-specific digestion of target DNA.
  • Engineered versions of naturally occurring meganucleases typically have extended DNA recognition sequences (for example, 14 to 40 bp).
  • TALENs because the DNA recognition and cleavage functions of meganucleases are intertwined in a single domain. Specialized methods of mutagenesis and high-throughput screening have been used to create novel meganuclease variants that recognize unique sequences and possess improved nuclease activity.
  • a vector provided herein can comprise any combination of a nucleic acid sequence encoding an Argonaute (non-limiting examples of Argonaute proteins include Thermus thermophilics Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium gregoryi Argonaute (NgAgo), homologs thereof, or modified versions thereof), and optionally, a sequence encoding DNA guide.
  • Argonaute proteins include Thermus thermophilics Argonaute (TtAgo), Pyrococcus furiosus Argonaute (PfAgo), Natronobacterium gregoryi Argonaute (NgAgo), homologs thereof, or modified versions thereof
  • TtAgo Thermus thermophilics Argonaute
  • PfAgo Pyrococcus furiosus Argonaute
  • Natronobacterium gregoryi Argonaute Natronobacterium gregoryi Argonaute
  • a vector provided herein can comprise any combination of a nucleic acid sequence encoding a RNA-guided Cas9 nuclease (non-limiting examples of RNA-guided nucleases include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl 2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, C
  • Cas9 nucleases are part of the adaptive immune system of bacteria and archaea, protecting them against invading nucleic acids such as viruses by cleaving the foreign DNA in a sequence-dependent manner.
  • the immunity is acquired by the integration of short fragments of the invading DNA known as spacers between two adjacent repeats at the proximal end of a CRISPR locus.
  • the CRISPR arrays, including the spacers, are transcribed during subsequent encounters with invasive DNA and are processed into small interfering CRISPR RNAs (crRNAs) approximately 40 nt in length, which combine with the tram-activating CRISPR RNA (tracrRNA) to activate and guide the Cas9 nuclease.
  • crRNAs small interfering CRISPR RNAs
  • a prerequisite for cleavage is the presence of a conserved protospacer-adjacent motif (PAM) downstream of the target DNA, which usually has the sequence 5-NGG-3 but less frequently NAG.
  • PAM protospacer-adjacent motif
  • Specificity is provided by the so-called “seed sequence” approximately 12 bases upstream of the PAM, which must match between the RNA and target DNA.
  • Cpf 1 acts in a similar manner to Cas9, but Cpfl does not require a tracrRNA.
  • a nucleic acid molecule provided herein comprises at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 site-specific enzyme target sites.
  • a vector or sequence of interest provided herein comprises a Cre/lox recombination site, a Flp/FRT recombination site, an endonuclease recognition site, a TALEN site, or any combination thereof.
  • a vector or sequence of interest provided herein comprises a Cre recombinase or a Flp recombination system.
  • a recombination system provided herein can act in cis.
  • a recombination system provided herein can act in trans.
  • a site-specific enzyme target site provided herein is at least 10, at least 20, at least 30, at least 40, at least 50, at least 75, at least 100, at least 125, at least 150, at least 200, at least 250, at least 300, at least 400, or at least 500 nucleotides.
  • the Flp- RJ site-directed recombination system comes from the 2 ⁇ plasmid from the baker's yeast Saccharomyces cerevisiae.
  • Flp recombinase flippase
  • FRT sites comprise 34 nucleotides.
  • Flp binds to the "arms" of the FRT sites (one arm is in reverse orientation) and cleaves the FRT site at either end of an intervening nucleic acid sequence. After cleavage, Flp recombines nucleic acid sequences between two FRT sites.
  • Cre-lox is a site-directed recombination system derived from the
  • Cre-lox can be used to invert a nucleic acid sequence, delete a nucleic acid sequence, or translocate a nucleic acid sequence.
  • Cre recombinase recombines a pair of lox nucleic acid sequences. Lox sites comprise 34 nucleotides, with the first and last 13 nucleotides (arms) being palindromic.
  • Cre recombinase protein binds to two lox sites on different nucleic acids and cleaves at the lox sites.
  • a lox site provided herein is a loxP, lox 2272, loxN, lox 511, lox 5171, lox71, lox66, M2, M3, M7, or Mi l site.
  • the instant disclosure provides a Rhizobiales cell comprising at least one vector that is capable of forming two essentially complementary T-strands.
  • the instant disclosure provides an Agrobacterium cell comprising at least one vector that is capable of forming two essentially complementary T-strands.
  • the instant disclosure provides a method of increasing the rate of site directed integration of a sequence of interest, comprising contacting a plant cell with a Rhizobiales cell capable of transforming the plant cell, where the Rhizobiales cell comprises at least one vector.
  • the instant disclosure provides a method of
  • transforming a plant cell comprising contacting the plant cell with a Rhizobiales cell capable of transforming the plant cell, where the Rhizobiales cell comprises at least one vector capable of forming two T-strands that are essentially complementary in at least a portion of a of the T-strands.
  • a vector disclosed herein comprises a first right border DNA sequence (RBI), a second right border DNA sequence (RB2), and at least one sequence of interest, where the RBI is positioned in the vector to initiate synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3 ' end of the first T-strand; and the RB2 is positioned in the vector to initiate synthesis of a second T-strand such that the sequence of interest is in the anti-sense orientation from the 5' to 3' end of the second T-strand, and where the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other.
  • RBI right border DNA sequence
  • RB2 second right border DNA sequence
  • a vector disclosed herein comprises a first right border
  • a vector disclosed herein comprises a first right border
  • DNA sequence (RBI), a second right border DNA sequence (RB2), at least one sequence of interest, and a left border DNA sequence (LB), where the vector is configured such that the RBI is paired with the LB, which are positioned in the vector to initiate (RBI) and terminate (LB) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the RB2 initiates synthesis of a second T- strand such that the sequence of interest is in an anti-sense orientation relative to the sequence of interest in the first T-strand, and where the two T-strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the sequence of interest.
  • a vector disclosed herein comprises a first right border
  • DNA sequence (RBI), a second right border DNA sequence (RB2), at least one sequence of interest, and a left border DNA sequence (LB), where the vector is configured such that the RBI initiates synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the RB2 is paired with the LB, which are positioned in the vector to initiate (RB2) and terminate (LB) synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation relative to the sequence of interest in the first T-strand, and where the two T-strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the sequence of interest.
  • a vector disclosed herein comprises a first sequence of interest and a second sequence of interest, where the first sequence of interest is essentially identical to the second sequence of interest; and the vector further comprises a first right border DNA sequence (RBI) with a first left border DNA sequence (LB 1) which are positioned in the vector to initiate (RBI) and terminate (LB1) synthesis of a first T-strand such that the first sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the vector further comprises a second right border DNA sequence (RB2) and a second left border DNA sequence (LB2) which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the second sequence of interest is in an anti-sense orientation relative to the first sequence of interest in the first T- strand, and where the two T-strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the first sequence of interest and the second sequence of interest.
  • RBI right border DNA sequence
  • a vector disclosed herein comprises a first sequence of interest and a second sequence of interest, where the first sequence of interest is essentially identical to the second sequence of interest; and the vector further comprises a first right border DNA sequence (RBI) with a left border DNA sequence (LB) which are positioned in the vector to initiate (RB I) and terminate (LB) synthesis of a first T-strand such that the first sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the vector further comprises a second right border DNA sequence (RB2) which initiates synthesis of a second T-strand such that the second sequence of interest is in an anti-sense orientation relative to the first sequence of interest in the first T-strand, and where the two T- strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the first sequence of interest and the second sequence of interest.
  • RBI right border DNA sequence
  • LB left border DNA sequence
  • RB2 second right border DNA sequence
  • a vector disclosed herein comprises a first sequence of interest and a second sequence of interest, where the first sequence of interest is essentially identical to the second sequence of interest; and the vector further comprises a first right border DNA sequence (RBI) which initiates synthesis of a first T-strand such that the first sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the vector further comprises a second right border DNA sequence (RB2) and a left border DNA sequence (LB) which are positioned in the vector to initiate (RB2) and terminate (LB) synthesis of a second T-strand such that the second sequence of interest is in an anti-sense orientation relative to the first sequence of interest in the first T-strand, and where the two T- strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the first sequence of interest and the second sequence of interest.
  • RBI right border DNA sequence
  • LB left border DNA sequence
  • a vector disclosed herein comprises a first sequence of interest and a second sequence of interest, where the first sequence of interest is essentially identical to the second sequence of interest; and the vector further comprises a first right border DNA sequence (RBI) to initiate synthesis of a first T-strand such that the first sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the vector further comprises a second right border DNA sequence (RB2) which initiates synthesis of a second T-strand such that the second sequence of interest is in an anti-sense orientation relative to the first sequence of interest in the first T-strand, and where the two T- strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the first sequence of interest and the second sequence of interest.
  • RBI right border DNA sequence
  • RB2 second right border DNA sequence
  • a Rhizobiales cell disclosed herein comprises at least a first vector and a second vector, where each vector comprises essentially identical sequences of interest, and where the first vector comprises a first right border DNA sequence (RBI) and a first left border DNA sequence (LB 1) which are positioned in the first vector to initiate (RBI) and terminate (LB 1) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the second vector comprises a second right border DNA sequence (RB2) and a second left border DNA Sequence (LB2) which are positioned in the second vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation relative to the sequence of interest in the first T-strand, and where the two T- strands resulting from initiation at RBI and RB2 are essentially complementary to each other in at least a portion of the sequence of interest.
  • the first vector comprises a first right border DNA sequence (RBI)
  • a Rhizobiales cell comprises at least a first vector and a second vector, where each vector comprises essentially identical sequences of interest, and where the first vector comprises a first right border DNA sequence (RBI) and a left border DNA sequence (LB) which are positioned in the first vector to initiate (RBI) and terminate (LB) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the second vector comprises a second right border DNA sequence (RB2) which initiates synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation relative to the sequence of interest in the first T-strand, and where the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other in at least a portion of the sequence of interest.
  • RBI right border DNA sequence
  • LB left border DNA sequence
  • a Rhizobiales cell comprises at least a first vector and a second vector, where each vector comprises essentially identical sequences of interest, and where the first vector comprises a first right border DNA sequence (RBI) which initiates synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the second vector comprises a second right border DNA sequence (RB2) and a left border DNA Sequence (LB) which are positioned in the second vector to initiate (RB2) and terminate (LB) synthesis of a second T- strand such that the sequence of interest is in an anti-sense orientation relative to the sequence of interest in the first T-strand, and where the two T-strands resulting from initiation at RB 1 and RB2 are essentially complementary to each other in at least a portion of the sequence of interest.
  • RBI right border DNA sequence
  • LB left border DNA Sequence
  • a Rhizobiales cell comprises at least a first vector and a second vector, where each vector comprises essentially identical sequences of interest, and where the first vector comprises a first right border DNA sequence (RB 1) which initiates synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the second vector comprises a second right border DNA sequence (RB2) which initiates synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation relative to the sequence of interest in the first T-strand, and where the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other in at least a portion of the sequence of interest.
  • RB 1 first right border DNA sequence
  • RB2 second right border DNA sequence
  • methods provided in the instant disclosure comprise two or more vectors, where the two or more vectors are in one Rhizobiales cell.
  • methods provided in the instant disclosure comprise two or more vectors, where the two or more vectors are in two or more Rhizobiales cells.
  • a first Rhizobiales cell comprises a first vector
  • a second Rhizobiales cell comprises a second vector.
  • the instant disclosure provides a method of increasing the rate of site directed integration of a sequence of interest, comprising contacting the plant cell with two or more Rhizobiales cells capable of transforming the plant cell, where the two or more Rhizobiales cells each contain one of at least two vectors capable of forming two essentially complementary T-strands.
  • the instant disclosure provides a method of transforming a plant cell, comprising contacting the plant cell with two or more Rhizobiales cells capable of transforming the plant cell, where the two or more Rhizobiales cells each contain one of at least two vectors capable of forming two essentially complementary T-strands.
  • the instant disclosure provides a method of increasing the rate of site directed integration of a sequence of interest, comprising contacting a plant cell with two or more Rhizobiales cells, where the two or more Rhizobiales cells each contain one of at least two vectors capable of forming two essentially complementary T-strands.
  • a first Rhizobiales cell and a second Rhizobiales cell contain at least a first vector and a second vector, respectively, where each vector comprises an essentially identical sequence of interest, and where the first vector comprises a first right border DNA sequence (RBI), and where the RBI is positioned in the vector to initiate synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the second vector comprises a second right border DNA sequence (RB2) which is positioned in the vector to initiate synthesis of a second T-strand such that the sequence of interest is in the anti-sense orientation relative to the sequence of interest in the first T-strand, and where the two T- strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the sequence of interest.
  • RBI right border DNA sequence
  • a first Rhizobiales cell and a second Rhizobiales cell contain at least a first vector and a second vector, respectively, where each vector comprises an essentially identical sequence of interest, and where the first vector comprises a first right border DNA sequence (RBI) and a first left border DNA sequence (LBl) which are positioned in the first vector to initiate (RBI) and terminate (LB l) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the second vector comprises a second right border DNA sequence (RB2) and a second left border DNA sequence (LB2) which are positioned in the second vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation from the 5' to 3' end of the second T- strand, and where the sequence of interest in the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other.
  • the first vector comprises a first right
  • the instant disclosure provides a method comprising a first Rhizobiales cell and a second Rhizobiales cell, where each Rhizobiales cell contains at least one of two vectors, where each vector comprises an essentially identical sequence of interest, and where the first vector comprises a first right border DNA sequence (RBI), and where the RBI is positioned in the vector to initiate synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the second vector comprises a second right border DNA sequence (RB2) which is positioned in the vector to initiate synthesis of a second T-strand such that the sequence of interest is in the anti-sense orientation relative to the sequence of interest in the first T-strand, and wherein the two T-strands resulting from initiation at RBI and RB2 are essentially complementary in at least a portion of the sequence of interest.
  • RBI right border DNA sequence
  • the instant disclosure provides a method comprising a first Rhizobiales cell and a second Rhizobiales cell, where each Rhizobiales cell contains at least one of two vectors, where each vector comprises an essentially identical sequence of interest, and where the first vector comprises a first right border DNA sequence (RBI) and a first left border DNA sequence (LB 1) which are positioned in the first vector to initiate (RB 1) and terminate (LB 1) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the second vector comprises a second right border DNA sequence (RB2) and a second left border DNA sequence (LB2) which are positioned in the second vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation from the 5' to 3' end of the second T-strand, and wherein the sequence of interest in the two T-strands resulting from initiation at RBI and RB2 are essentially complementary
  • the instant disclosure provides a method comprising a first Rhizobiales cell and a second Rhizobiales cell, where each Rhizobiales cell contains at least one of two vectors, where each vector comprises an essentially identical sequence of interest, and where the first vector comprises a first right border DNA sequence (RBI) and a left border DNA sequence (LB) which are positioned in the first vector to initiate (RBI) and terminate (LB) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the second vector comprises a second right border DNA sequence (RB2) to initiate synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation from the 5' to 3' end of the second T- strand, and wherein the sequence of interest in the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other.
  • RBI right border DNA sequence
  • LB left border DNA sequence
  • the instant disclosure provides a method comprising a first Rhizobiales cell and a second Rhizobiales cell, where each Rhizobiales cell contains at least one of two vectors, where each vector comprises an essentially identical sequence of interest, and where the first vector comprises a first right border DNA sequence (RBI) initiate synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the second vector comprises a second right border DNA sequence (RB2) and a left border DNA sequence (LB) which are positioned in the second vector to initiate (RB2) and terminate (LB) synthesis of a second T- strand such that the sequence of interest is in an anti-sense orientation from the 5' to 3' end of the second T-strand, and wherein the sequence of interest in the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other.
  • RBI right border DNA sequence
  • LB left border DNA sequence
  • At least one vector disclosed herein comprises an RB and a LB and where the vector further comprises between the RB and LB: (i) a first sequence of interest in a sense orientation relative to the RB, (ii) a spacer, and (iii) a second sequence of interest in an anti-sense orientation relative to the RB, where the first sequence of interest and the second sequence of interest are essentially complementary and after synthesis of the T-strand anneal to form a double-stranded DNA.
  • the integration of at least part of the sequence of interest in a vector results in a point mutation, an insertion, a deletion, an inversion, increased
  • transcription of an endogenous genomic locus decreased transcription of an endogenous genomic locus, altered protein activity, increased transcription of the sequence of interest, decreased transcription of the integrated sequence of interest, or a combination thereof.
  • the sequence of interest in the vector further comprises at least one, at least two, at least three, at least four, at leave five, at least six, at least seven, at least eight, at least nine, or at least ten site-specific enzyme target sites.
  • site-specific enzyme target site include, but are not limited to, a Cre/lox recombination site, a Flp/FRT recombination site, a endonuclease recognition site, and a TALEN site.
  • the Rhizobiales cell is selected from an Agrobacterium spp., a Bradyrhizobium spp., & Mesorhizobium spp., an Ochrobactrum spp., & Phyllobacterium spp., a Rhizobium spp., Sinorhizobium spp, and an Ensifer spp.
  • the Rhizobiales cell is an Agrobacterium cell.
  • the Rhizobiales cell is an Agrobacterium tumefaciens cell.
  • the Rhizobiales cell further contains a vector comprising one or more gene expression cassettes with sequence encoding a cell factor that functions to increase DNA repair, one or more Agrobacterium Ti plasmid vir genes, one or more selectable marker genes, an origin of replication, or any combination thereof.
  • the instant disclosure provides a method of increasing the rate of site-directed integration of a sequence of interest in a plant genome, comprising contacting at least one plant cell on a co-culture medium for at least two days, at least 3 days, at least 4 days or at least 5 days with a Rhizobiales cell capable of transforming the plant cell, wherein the Rhizobiales cell comprises at least one vector described in the instant disclosure.
  • the instant disclosure provides a method of transforming a plant genome, comprising contacting at least one plant cell on a co-culture medium for at least two days, at least 3 days, at least 4 days, or at least 5 days with at least one Rhizobiales cell capable of transforming the plant cell, where the Rhizobiales cell comprises at least one vector capable of forming two essentially complementary T-strands.
  • the contacting comprises co-culturing the plant cell with a
  • Rhizobiales cell for at least three days, at least four days, at least five days, at least six days, at least seven days, at least eight days, at least nine days, or at least ten days.
  • the contacting comprises co-culturing the plant cell with a Rhizobiales cell for at least 48 hours, at least 49 hours, at least 50 hours, at least 51 hours, at least 52 hours, at least 53 hours, at least 54 hours, at least 55 hours, at least 56 hours, at least 57 hours, at least 58 hours, at least 59 hours, at least 60 hours, at least 61 hours, at least 62 hours, at least 63 hours, at least 64 hours, at least 65 hours, at least 66 hours, at least 67 hours, at least 68 hours, at least 69 hours, at least 70 hours, at least 71 hours, at least 72 hours, at least 73 hours, at least 74 hours, at least 75 hours, at least 76 hours, at least 77 hours, at least 78 hours, at least 79 hours, at least 80 hours,
  • a Rhizobiales cell is selected from the group consisting of an Agrobacterium spp. cell, a Bradyrhizobium spp. cell, a Mesorhizobium spp. cell, an Ochrobactrum spp. cell, a Phyllobacterium spp. cell, a Rhizobium spp. cell, and a Sinorhizobium spp. cell.
  • an Agrobacterium spp. cell is selected from the group consisting of an Agrobacterium tumefaciens cell and an Agrobacterium rhizogenes cell.
  • the method further comprises detecting the integration of at least a fragment of the sequence of interest of the vector in the at least one plant cell.
  • the at least one fragment of the sequence of interest in the vector is integrated into the plant genome by HR.
  • the at least one fragment of the sequence of interest in the vector is integrated into the plant genome by HEJ.
  • the method also comprises selecting the plant call based on the presence of the at least one fragment of the sequence of interest in the vector integrated into the plant genome. In a further aspect, the method further comprises regenerating a transgenic plant form the selected plant cell.
  • a method or system for site-specific modification of a target nucleic acid sequence provided herein involves homologous recombination. In another aspect, a method or system for site-specific modification of a target nucleic acid sequence provided herein involves non-homologous end joining.
  • a method or system for site-specific modification of a target nucleic acid sequence comprises non-homologous end joining that further comprises the introduction of an insertion and/or deletion of one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, ten or more, twenty or more, fifty or more, or one hundred or more nucleotides into the target nucleic acid sequence.
  • microprojectile bombardment with particles coated with recombinant DNA are found in US Patents 5,015,580 (soybean); 5,550,318 (corn); 5,538,880 (corn); 5,914,451 (soybean);
  • a method provided herein stably transforms a plant cell.
  • a method provided herein transiently transforms a plant cell.
  • a method of transforming a plant cell provided herein comprises a biolistic transformation or a bacteria-mediated transformation.
  • a method of transforming a plant cell provided herein comprises bacteria-mediated transformation that further comprises contacting the plant cell with a Rhizobiales cell, where the Rhizobiales cell is capable of transforming the plant cell.
  • Transformation methods to provide transgenic plant cells and transgenic plants containing stably integrated nucleic acid molecules provided herein are preferably practiced in tissue culture on media and in a controlled environment.
  • Media refers to the numerous nutrient mixtures that are used to grow cells in vitro, that is, outside of the intact living organism.
  • this disclosure provides plant cells that are not reproductive material and do not mediate the natural reproduction of the plant. In another aspect, this disclosure also provides plant cells that are reproductive material and mediate the natural reproduction of the plant. In another aspect, this disclosure provides plant cells that cannot maintain themselves via photosynthesis. In another aspect, this disclosure provides somatic plant cells. Somatic cells, contrary to germline cells, do not mediate plant reproduction.
  • Recipient cell targets for transformation include, but are not limited to, a seed cell, a fruit cell, a leaf cell, a cotyledon cell, a hypocotyl cell, a meristem cell, an embryo cell, an endosperm cell, a root cell, a shoot cell, a stem cell, a pod cell, a flower cell, an
  • this disclosure provides a plant chloroplast.
  • this disclosure provides an epidermal cell, a stomata cell, a trichome cell, a root hair cell, a storage root cell, or a tuber cell.
  • this disclosure provides a protoplast.
  • this disclosure provides a plant callus cell. Any cell from which a fertile plant can be regenerated is contemplated as a useful recipient cell for practice of this disclosure.
  • Callus can be initiated from various tissue sources, including, but not limited to, immature embryos or parts of embryos, seedling apical meristems, microspores, and the like. Those cells which are capable of proliferating as callus can serve as recipient cells for transformation. Practical
  • transgenic plants of this disclosure e.g., various media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants
  • transformation methods and materials for making transgenic plants of this disclosure e.g., various media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants
  • a plant cell provided herein is selected from the group consisting of an Acacia cell, an alfalfa cell, an aneth cell, an apple cell, an apricot cell, an artichoke cell, an arugula cell, an asparagus cell, an avocado cell, a banana cell, a barley cell, a bean cell, a beet cell, a blackberry cell, a blueberry cell, a broccoli cell, a Brussels sprout cell, a cabbage cell, a canola cell, a cantaloupe cell, a carrot cell, a cassava cell, a cauliflower cell, a celery cell, a Chinese cabbage cell, a cherry cell, a cilantro cell, a citrus cell, a
  • Clementine cell a coffee cell, a corn cell, a cotton cell, a cucumber cell, a Douglas fir cell, an eggplant cell, an endive cell, an escarole cell, an eucalyptus cell, a fennel cell, a fig cell, a forest tree cell, a gourd cell, a grape cell, a grapefruit cell, a honey dew cell, a jicama cell, kiwifruit cell, a lettuce cell, a leek cell, a lemon cell, a lime cell, a Loblolly pine cell, a mango cell, a maple tree cell, a melon cell, a mushroom cell, a nectarine cell, a nut cell, an oat cell, an okra cell, an onion cell, an orange cell, an ornamental plant cell, a papaya cell, a parsley cell, a pea cell, a peach cell, a peanut cell, a pear cell, a pepper cell,
  • a plant cell provided herein is selected from the group consisting of a corn immature embryo cell, a corn mature embryo cell, a corn seed cell, a soybean immature embryo cell, a soybean mature embryo cell, a soybean seed cell, a canola immature embryo cell, a canola mature embryo cell, a canola seed cell, a cotton immature embryo cell, a cotton mature embryo cell, a cotton seed cell, a wheat immature embryo cell, a wheat mature embryo cell, a wheat seed cell, a sugarcane immature embryo cell, a sugarcane mature embryo cell, a sugarcane seed cell.
  • transformation of a plant cell is performed by an Agrobacterium or other Rhizobiales-mediated method (U.S. Pat. Nos. 6,265,638, 5,731,179; U.S. Patent Application Publications US2005/0183170; 2003110532).
  • the polynucleotide sequences that can be transferred into a plant cell provided herein can be present on one recombination vector in one bacterial strain being utilized for transformation.
  • the polynucleotide sequences provided herein can be present on separate recombination vectors in one bacterial strain.
  • the polynucleotide sequences provided herein can be found in separate bacterial cells or strains used together for transformation.
  • the DNA constructs used for transformation in the methods of present disclosure generally also contain the plasmid backbone DNA segments that provide replication function and antibiotic selection in bacterial cells, for example, an Escherichia coli origin of replication such as ori322, an Agrobacterium origin of replication such as oriV or oriRi, and a coding region for a selectable marker such as Spec/Strp that encodes for Tn7 aminoglycoside adenyltransferase (aadA) conferring resistance to spectinomycin or streptomycin, or a gentamicin (Gm, Gent) selectable marker gene.
  • aadA Tn7 aminoglycoside adenyltransferase
  • Gm, Gent gentamicin
  • the host bacterial strain is often Agrobacterium tumefaciens ABI, C58, LBA4404, AGLO, AGL1, EHA101, or EHA105 carrying a plasmid having a transfer function for the expression unit.
  • Other strains known to those skilled in the art of plant transformation can function in this disclosure.
  • assays include, for example, molecular biological assays (e.g., Southern and northern blotting, PCRTM); biochemical assays, such as detecting the presence of a protein product (e.g., by immunological means (ELISAs and western blots), or by enzymatic function (e.g., GUS assay)); pollen histochemistry; plant part assays, (e.g., leaf or root assays); and also, by analyzing the phenotype of the whole regenerated plant.
  • molecular biological assays e.g., Southern and northern blotting, PCRTM
  • biochemical assays such as detecting the presence of a protein product (e.g., by immunological means (ELISAs and western blots), or by enzymatic function (e.g., GUS assay)); pollen histochemistry; plant part assays, (e.g., leaf or root assays); and also, by analyzing the phenotype of the
  • the instant disclosure also provides a transgenic plant cell comprising a sequence of interest integrated into a genome of the plant cell according to the methods disclosed herein. Also provided is a transgenic plant produced by the methods disclosed herein.
  • Both control vectors contain a sequence of interest comprising three expression cassettes: an expression cassette encoding a gene (CP4-EPSPS) to confer tolerance to the herbicide glyphosate positioned between a left homology arm and a right homology arm; and two expression cassettes each encoding half of a TALEN pair.
  • TALENs were obtained from Life Technologies.
  • the first control vector comprised a RB DNA sequence, the sequence of interest comprising the three expression cassettes, and a LB DNA sequence, as illustrated in Figure 4 A.
  • the second control vector was the same as the first control vector except that there was no LB DNA sequence. These two control vectors were used in Example 9, below.
  • the vector was constructed to contain a sequence of interest comprising three expression cassettes: one expression cassette encoding a gene (CP4-EPSPS) to confer tolerance to the herbicide glyphosate positioned between a left homology arm and a right homology arm; and two expression cassettes each encoding half of a TALEN pair; and two RB DNA sequences (RBI and RB2).
  • CP4-EPSPS CP4-EPSPS
  • the vector configuration comprised the first RB DNA sequence (RBI) positioned in the vector to initiate synthesis of a first T-strand comprising the three expression cassettes (CP4-EPSPS and two TALENs); and the second RB DNA sequence (RB2) was positioned in the vector to initiate synthesis of a second T- strand comprising the three expression cassettes (CP4-EPSPS and two TALENs) such that the second T-strand was in an anti-sense orientation relative to the first T-strand.
  • the two T- strands resulting from initiation at RBI and RB2 are essentially complementary to each other in at least a portion of the sequence of interest.
  • a vector comprising two RB DNA sequences, and one LB DNA sequence was created for Agrobacterium-mediated transformation ( Figure 4C).
  • the vector was constructed to contain a sequence of interest comprising three expression cassettes: one expression cassette encoding a gene (CP4-EPSPS) to confer tolerance to the herbicide glyphosate positioned between a left homology arm and a right homology arm; and two expression cassettes each encoding half of a TALEN pair; and two right border DNA sequences (RBI and RB2), and one LB DNA sequence (LB 1) paired with the first RB DNA sequence (RBI).
  • the vector configuration comprised the first RB DNA sequence (RBI) positioned in the vector to initiate synthesis of a first T-strand comprising the three expression cassettes (CP4-EPSPS and two TALENs) and terminate synthesis at the first LB DNA sequence (LB1) (paired with RBI); and the second RB DNA sequence (RB2) was positioned in the vector to initiate synthesis of a second T-strand comprising the three expression cassettes (CP4-EPSPS and two TALENs) such that the second T-strand was in an anti-sense orientation relative to the first T-strand.
  • This vector was used in Example 9 below.
  • the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other in at least a portion of the sequence of interest.
  • a vector comprising at least one sequence of interest, two RB DNA
  • the vector configuration comprises a first RB DNA sequence (RBI) paired with a first LB DNA sequence (LB1) which are positioned in the vector to initiate (RBI) and terminate (LB1) synthesis of a first T- strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the second RB DNA sequence (RB2) is paired with a second LB DNA sequence (LB2) which are positioned in the vector to initiate synthesis of a second T-strand such that the sequence of interest is in an anti-sense orientation relative to the sequence of interest in the first T-strand.
  • the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other in at least a portion of the sequence of interest.
  • Example 5 Construct of a vector with two RB DNA sequences and two essentially identical sequences of interest
  • a vector comprising two essentially identical sequences of interest, at least two RB DNA sequences, and optional one or more LB DNA sequences is created for
  • the vector configuration comprises a first RB DNA sequence (RBI) with an optional first LB DNA sequence (LBl) which are positioned in the vector to initiate (RBI) and terminate (LBl) synthesis of a first T-strand such that the first sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the vector configuration further comprises a second RB DNA sequence (RB2) and an optional second LB DNA sequence (LB2) which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the second sequence of interest is in an anti-sense orientation relative to the sequence of interest in the first T-strand.
  • the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other in at least a portion of the sequence of interest.
  • the two vector configurations comprise essentially identical sequences of interest, where the first vector configuration comprises a first RB DNA sequence (RBI) and an optional first LB DNA sequence (LB 1 ) which are positioned in the first vector to initiate (RB 1 ) and terminate (LBl) synthesis of a first T-strand such that the sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the second vector configuration comprises a second RB DNA sequence (RB2) and an optional second LB DNA sequence (LB2) which are positioned in the second vector to initiate (RB2) and terminate (LB2) synthesis of a second T- strand such that the sequence of interest is in an anti-sense orientation relative to the sequence of interest in the first T-strand.
  • the first vector configuration comprises a first RB DNA sequence (RBI) and an optional first LB DNA sequence (LB 1 ) which are positioned in the first vector to initiate (RB 1 ) and terminate (LBl) synthesis of a first T-strand such that the sequence of interest is in the sense orientation
  • the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other in at least a portion of the sequence of interest.
  • the two vectors may be in the same Agrobacterium cell, or the two vectors may be in different Agrobacterium cells.
  • Example 7. Construct of a vector with two different sequences of interest and three or more RB DNA sequences
  • a vector comprising a first sequence of interest, a second sequence of interest different from the first sequence of interest, three or more RB DNA sequences, and optional one or more LB DNA sequences is created for Agrobacterium-mediated transformation.
  • a vector configuration is presented in
  • Figure 2C where the vector configuration comprises a first RB DNA sequence (RBI) and a first LB DNA sequence (LBl) which are positioned in the vector to initiate (RBI) and terminate (LB1) synthesis of a first T-strand such that the first sequence of interest is in the sense orientation from the 5' to 3' end of the first T-strand; and the vector configuration further comprises a second RB DNA sequence (RB2) and a second LB DNA sequence (LB2) which are positioned in the vector to initiate (RB2) and terminate (LB2) synthesis of a second T-strand such that the first sequence of interest is in an anti-sense orientation relative to the sequence of interest in the first T-strand.
  • RBI RB DNA sequence
  • LBl first LB DNA sequence
  • the two T-strands resulting from initiation at RBI and RB2 are essentially complementary to each other in at least a portion of the sequence of interest.
  • the vector configuration further comprises a third RB DNA sequence (RB3) and a third LB DNA sequence (LB3) which are positioned in the vector to initiate (RB3) and terminate (LB3) synthesis of a third T-strand such that the second sequence of interest is in the sense orientation from the 5' to 3' end of the third T-strand; and the vector configuration further comprises a fourth RB DNA sequence (RB4) and a fourth LB DNA sequence (LB4) which are positioned in the vector to initiate (RB4) and terminate (LB4) synthesis of a fourth T-strand such that the second sequence of interest is in an anti-sense orientation relative to the sequence of interest in the third T-strand.
  • the two T-strands resulting from initiation at RB3 and RB4 are essentially complementary to each other in at least a portion of the sequence of interest.
  • Example 8 Construct of a vector with a RB DNA sequence, a LB DNA sequence, and two sequences of interest linked by a spacer
  • a vector is provided for Agrobacterium-mediated transformation where the vector configuration comprises a RB DNA sequence and a LB DNA sequence and where the vector further comprises between the RB and LB DNA sequences: (i) a first sequence of interest in a sense orientation relative to the RB DNA sequence, (ii) a spacer, and (iii) a second sequence of interest in an anti-sense orientation relative to the first sequence of interest.
  • the two sequences of interest after synthesis of the T-strand, form a partially double stranded hairpin structure due to complementary base pairing.
  • Figure 3 A one non-limiting example of such a vector configuration is presented in Figure 3 A.
  • Example 9 Increased frequency of site-directed integration by Agrobacterium-m diat d transformation
  • L7 A locus termed L7 was identified as occurring only once in a corn genome. A site was selected within the L7 locus for insertion of a transgene by site-directed
  • TALENs To facilitate site-directed recombination a pair of TALENs was engineered to introduce a double-strand break at a specific site within the L7 locus.
  • Vectors made according to Examples 1-3 were transformed into immature corn embryos. The vectors each comprised a sequence of interest containing three expression cassettes: two expression cassettes for expression of each one of the TALEN pair and an expression cassette containing a CP4-EPSPS transgene positioned between a left homology arm and a right homology arm.
  • Agrobacterium containing one of the four vectors for 3 days then moved to callus-induction medium containing 0.1 mM glyphosate as a selection agent. Approximately 300 regenerated plants (R0 events) were selected for each vector, transferred to plugs, and grown in a greenhouse. Genomic DNA was extracted from R0 leaf tissue after one week of greenhouse growth, and individual plants were molecularly assessed with real time PCR for (a) the presence of the transgene (CP4-EPSPS) copy number, and (b) targeting sequence copy number.
  • CP4-EPSPS transgene
  • PCR with genomic DNA from of selected R0 plants was used to identify individual plants comprising CP4-EPSPS cassette insertions at the L7 target site.
  • PCR primers were designed such that a product was only produced when the CP4-EPSPS cassette inserted into the L7 targeted region of the corn genome; one PCR primer was located in genomic DNA flanking the targeted region, and one PCR primer was located within the CP4- EPSPS cassette.
  • Two sets of PCR primers were used, one positioned on the 5' end of the CP4-EPSPS cassette and one positioned on the 3' end of the CP4-EPSPS cassette.
  • Figure 5 shows the positions of the primers used to identify CP4-EPSPS cassette insertions in the L7 locus.
  • the 5' set of PCR primers amplifies a 2676 bp product when the CP4-EPSPS cassette is inserted in the targeted L7 locus; no product is produced in wild type genomic DNA or if the CP4-EPSPS cassette is not inserted into the L7 target site.
  • the 3' set of PCR primers amplifies a 2282 bp product when the CP4-EPSPS is inserted in the targeted L7 locus; no product is produced in wild type genomic DNA or if the CP4-EPSPS cassette is not inserted into the L7 target site.
  • the products were resolved on an agarose gel to identify plants with the correct sized bands for both the 5' end and the 3' end of the CP4-EPSPS cassette.
  • plants with at least one positive PCR result for site directed insertion of the CP4-EPSPS cassette were selected for Southern blot analysis.
  • Genomic DNA extracted from the plant was digested with the restriction endonuclease Kpnl.
  • Digestion with Kpnl produces a 9.6 kb fragment when the CP4-EPSPS cassette is integrated into the L7 target site (see Figure 5).
  • Digestion with Kpnl produces a 5.1 kb DNA fragment corresponding to the L7 genomic locus, for example in the wild-type non-transformed plant or a plant where the CP4-EPSPS cassette has not integrated into the L7 locus (see Figure 5).
  • the Southern blot was probed with a "left flank probe" as shown in Figure 5.
  • TALEN- mediated site-directed integration using Agrobacterium vectors comprising either two RB DNA sequences (vector C) or two RB DNA sequences and one LB DNA sequence (vector D) had superior efficacy compared to standard Agrobacterium vectors comprising either one RB DNA sequence and one LB DNA sequence (vector A), or one RB DNA sequence (vector B).
  • the data indicate the unexpected observation of an increased frequency of site-directed integration by NHEJ or HR when the vector comprised two RB DNA sequences positioned to generate essentially complementary T-strands.
  • the data further indicated an increased number of events generated by HR when the vector comprised two RB DNA sequences positioned to generate essentially complementary T-strands (see Table 2).
  • Table 2 Summary of border configurations and site-directed integration (SDI) frequencies. Pos., positive; L, left; R, right; HEJ, non-homologous end-joining; HR, homologous recombination.
  • Example 10 Retargeting a gene to a pre-existing lox site
  • a vector with two RB DNA sequences is used to facilitate site-directed integration of a sequence of interest into a pre-existing recombination site (e.g. a lox site) in genomic DNA.
  • the vector is created comprising, from 5' to 3 ', a first right RB DNA sequence (RB I); P-35S-crtB; a first lox site; a gene; a first recombination site (optional); a marker gene to select for transformants; a second recombination site (optional); a second lox site; P-DaMV-Cre; and a second RB DNA sequence (RB2).
  • the RB I and RB2 are positioned in the vector to generate essentially complementary T-strands.
  • P-35S-crtB is a non-lethal, constitutively expressed phytoene synthase expression cassette that inhibits shoot elongation in cells where non-targeted recombination occurs.
  • P-DaMV-Cre is a constitutively expressed Cre-recombinase that promotes recombination of the intervening DNA construct with the targeted genomic lox site.
  • the lox sites of the intervening DNA recombine with a pre-existing genomic DNA lox site and insert into the genomic DNA.
  • a target after transformation and recombination contains only the regions of the intervening DNA construct between the two lox sites (in this example, a gene; a first recombination site; a marker gene; and a second recombination site).
  • Optional recombination sites are used to remove the marker gene in a future recombination event.
  • Agrobacterium containing a vector is typically incubated with the immature corn embryos for 12-16 hours.
  • the unexpected observation detailed herein was that extended co-culture of the Agrobacterium containing a vector with the immature corn embryos resulted in a higher percentage of targeted integrations of the sequence of interest contained in the vector while not significantly affecting transformation frequency.
  • Vector A ( Figure 4A, Example 1) was used for Agrobacterium-mediated transformation using standard protocols except that the co-culture time was varied. About 3000 immature corn embryos were co-cultured for each of 1 day, 2 days, or 3 days. After transformation, the transformants were moved to callus-induction medium containing 0.1 mM glyphosate as a selection agent. R0 plants surviving glyphosate selection were transferred to plugs and grown in a greenhouse.
  • Example 9 The PCR protocol and primers described in Example 9 were used with the genomic DNA from selected R0 plants from the extended co-culture protocol to identify individual plants comprising targeted integration of the CP4-EPSPS cassette. As described in Example 9, and illustrated in Figure 5, two sets of PCR primers were used to detect targeted integration of the CP4-EPSPS cassette at the L7 locus. To further verify whether the junction sequences at the targeted sites were perfect (e.g., HR) or imperfect (e.g., NHEJ), the PCR products were sequenced. Table 5 shows that the highest number of positive PCR results for either the left flank, the right flank, or both the left and right flanks was with the 3 -day co- culture.
  • HR perfect
  • NHEJ imperfect
  • Example 12 Increased frequency of site-directed integration of a Sequence of Interest flanked by RBs and optional LBs.
  • CP4-EPSPS CP4-EPSPS
  • a pair of TALENs was engineered to introduce a double-strand break at a specific site within the L7 locus.
  • a control vector (Vector 6A) was created for Agrobacterium-mediated transformation (see Figure 6A).
  • the control vector comprised a first sequence of interest comprising an expression cassette encoding CP4-EPSPS positioned between a first RB DNA sequence (RBI) and a first LB DNA sequence (LB1) such that T-strand synthesis initiated at RBI and terminated at LB1.
  • the vector further comprised a second sequence of interest comprising two expression cassettes each encoding half of a TALEN pair positioned between a second RB DNA sequence (RB2) and a second LB DNA sequence (LB2) such that T-strand synthesis initiated at RB2 and terminated at LB2.
  • TALENs were obtained from Life
  • a second vector was created for Agrobacterium-mediated transformation (see Figure 6B).
  • This vector comprised a first sequence of interest comprising an expression cassette encoding CP4-EPSPS positioned between two RB DNA sequences and two LB DNA sequences.
  • the vector further comprised a second sequence of interest comprising two expression cassettes each encoding half of a TALEN pair.
  • the vector configuration comprised a first RB DNA sequence (RBI) paired with a first LB DNA sequence (LB 1) which were positioned in the vector to initiate (RBI) and terminate (LB 1) synthesis of a first T-strand comprising CP4-EPSPS in an anti-sense orientation from the 5' to 3 ' end of the first T-strand; and the second RB DNA sequence (RB2) was paired with a second LB DNA sequence (LB2) which were positioned in the vector to initiate synthesis of a second T-strand such that CP4-EPSPS was in sense orientation from the 5' to 3' end of the second T-strand.
  • RBI was also positioned such that read through of the LB1 would result in the synthesis of a T-strand comprising both the CP4-EPSPS cassette and the two TALEN cassettes.
  • the T-strands resulting from initiation at RBI and RB2 were essentially complementary to each other in at least a portion of the first sequence of interest.
  • callus-induction medium containing 0.1 mM glyphosate as a selection agent.
  • genomic DNA was isolated from selected R0 plants and PCR assays were carried out to identify individual plants comprising CP4-EPSPS cassette insertions at the L7 target site.
  • PCR primers were designed such that a product was only produced when the CP4-EPSPS cassette inserted into the L7 target region of the corn genome.
  • PCR protocol and primers described in Example 9, and illustrated in Figure 5 were used to identify individual plants comprising targeted integration of the CP4-EPSPS cassette.
  • the PCR products were resolved on agarose gels to identify plants with the correct sized bands for both the 5' end and the 3 ' end of the CP4- EPSPS cassette.
  • Vector 6A of the 180 RO plants that were screened by PCR, none were positive for the left flank and 2 were positive for the right flank.
  • vector 6B of the 371 RO plants that were screened by PCR, 2 were positive for the left flank and 10 were positive for the right flank.
  • Example 13 Increased frequency of site-directed integration of a Sequence of interest flanked by RBs and no LBs.
  • CP4-EPSPS CP4-EPSPS
  • a pair of TALENs was engineered to introduce a double-strand break at a specific site within the L7 locus.
  • a first vector was created for Agrobacterium-mediated transformation.
  • the vector comprised a first sequence of interest comprising an expression cassette encoding CP4-EPSPS with a TALEN target site (TS) positioned 5' to the cassette.
  • the first sequence of interest was flanked by a first RB DNA sequence (RBI) and a second RB DNA sequence (RB2) positioned such that T-strand synthesis initiated at both RBI and RB2 and the resulting T-strands were essentially complementary to each other in at least a portion of the first sequence of interest.
  • RBI first RB DNA sequence
  • RB2 second RB DNA sequence
  • the vector further comprised a second sequence of interest comprising two expression cassettes each encoding half of a TALEN pair, where the second sequence of interest was positioned adjacent to a third RB DNA sequence (RB3) so as to initiate synthesis of a third T-strand that comprised the two TALEN cassettes.
  • TALENs were obtained from Life Technologies
  • a second vector was created for Agrobacterium-mediated transformation.
  • the vector comprised a first sequence of interest comprising an expression cassette encoding CP4-EPSPS flanked by TALEN target sites (TS). Additionally, the first sequence of interest was positioned between a first RB DNA sequence (RB 1) and a second RB DNA sequence (RB2) such that T-strand synthesis initiated at both RBI and RB2 and the resulting T-strands were essentially complementary to each other in at least a portion of the first sequence of interest.
  • RB 1 first RB DNA sequence
  • RB2 second RB DNA sequence
  • the vector further comprised a second sequence of interest comprising two expression cassettes each encoding half of a TALEN pair positioned adjacent to a third RB DNA sequence (RB3) so as to initiate synthesis of a third T-strand that comprised the two TALEN cassettes.
  • TALENs were obtained from Life Technologies.
  • a third vector (Vector 7C) was created for Agrobacterium-mediated transformation.
  • the vector configuration is presented in Figure 7C.
  • the vector comprised a first sequence of interest comprising an expression cassette encoding CP4-EPSPS positioned between a left homology arm and a right homology arm.
  • the first sequence of interest was positioned between a first RB DNA sequence (RBI) and a second RB DNA sequence (RB2) such that T-strand synthesis initiated at both RBI and RB2 and the resulting T-strands were essentially complementary to each other in at least a portion of the first sequence of interest.
  • the vector further comprised a second sequence of interest comprising two expression cassettes each encoding half of a TALEN pair positioned adjacent to a third RB DNA sequence (RB3) so as to initiate synthesis of a third T-strand that comprised the two TALEN cassettes.
  • TALENs were obtained from Life Technologies.
  • Agrobacterium containing one of the three vectors for 3 days then moved to callus-induction medium containing 0.1 mM glyphosate as a selection agent. Approximately 200 regenerated plants (R0 events) were selected for each vector, transferred to plugs, and grown in a greenhouse.
  • genomic DNA was isolated from selected R0 plants and PCR assays were carried out to identify individual plants comprising CP4-EPSPS cassette insertions at the L7 target site.
  • PCR primers were designed such that a product was only produced when the CP4-EPSPS cassette inserted into the targeted L7 region of the corn genome.
  • PCR protocol and primers described in Example 9, and illustrated in Figure 5 were used to identify individual plants comprising targeted integration of the CP4-EPSPS cassette.
  • the products were resolved on agarose gels to identify plants with the correct sized bands for both the 5' end and the 3' end of the CP4-EPSPS cassette.
  • Vector 7A of the 226 R0 plants that were screened by PCR, one was positive for the left flank and 4 were positive for the right flank.
  • Vector 7B of the 292 R0 plants that were screened by PCR, 2 were positive for the left flank and 26 were positive for the right flank.
  • One plant was positive for both right and left PCR.
  • Vector 7C of the 241 R0 plants that were screened by PCR, 7 were positive for the left flank and 7 were positive for the right flank. Four plants were positive for both right and left flank PCR products.
  • the data indicate that TALEN-mediated site-directed integration (SDI) using an Agrobacterium vector comprising two TALEN target sites and two RB DNA sequences flanking a cargo sequence of interest (Vector 7B) had superior efficacy compared to an Agrobacterium vector comprising only 1 target site and two RB DNA sequences flanking the cargo sequence (Vector 7A).
  • the data further indicate an increased number of full integration events generated by FIR when the vector comprised a cargo sequence flanked by homology arms and two RB DNA sequences (Vector 7C).
  • Agrobacterium comprising a transformation vector is typically incubated with immature corn embryos for 12-16 hours.
  • the unexpected observation detailed herein was that prolonging the co-culture time of the Agrobacterium comprising a vector with immature corn embryos to three days resulted in a higher number of events that had targeted integration of the sequence of interest contained in the vector.
  • a vector depicted in Figure 4A and Example 1 was used for Agrobacterium- mediated transformation using standard protocols except that the co-culture time was varied. Explants were co-cultured with Agrobacterium for 1, 3, 5, or 7 days. Transformation of immature embryos was conducted on eight different experimental dates. One and three day co-culture treatments were conducted on alternating dates (a total of 4 experimental treatment dates each), and an average of 1086 and 1132 explants were treated per experiment, respectively. Five and seven day treatments were conducted on each of the eight
  • PCR protocol and primers described in Example 9 were used with the genomic DNA from RO plants from the extended co-culture protocol to identify individual plants comprising targeted integration of the CP4- EPSPS cassette. As described in Example 9, and illustrated in Figure 5, two sets of PCR primers were used to detect targeted integration of the CP4-EPSPS cassette at the L7 locus.
  • the number of site directed integration events (determined by positive flank PCR results for either or both flanks) detected per experimental treatment was divided by the number of explants recovered to estimate the SDI frequencies.
  • the data were analyzed using a mixed linear model. Table 8 shows that the highest number of positive PCR results for either the left flank, the right flank, or both the left and right flanks was with the 3 -day co-culture.

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Abstract

La présente invention concerne le domaine de la transformation de plantes. L'invention concerne notamment des procédés permettant d'augmenter le taux d'intégration dirigée d'une séquence d'intérêt dans des plantes. L'invention concerne également des procédés permettant d'augmenter l'efficacité de la transformation de plantes médiée par les rhizobiums.
EP17821073.8A 2016-06-28 2017-06-27 Procédés et compositions destinés à être utilisés dans la modification du génome de plantes Pending EP3475427A4 (fr)

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